in situ atr-ftir spectroscopy on the deposition and protein interaction of polycation/alginate...
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RESEARCH
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DOI: 10.1002/adem.201080059In Situ ATR-FTIR Spectroscopy on the Deposition and ProteinInteraction of Polycation/Alginate Multilayers**
By Martin Muller*, Bernhard Torger and Bernd Keßler
We report in situ ATR-FTIR spectroscopy studies of the deposition of biocompatible polyelectrolytemultilayers (PEMs) consisting of the polycation chitosan (CHT) or poly(ethyleneimine) (PEI) and thepolyanion sodium alginate (ALG) and their interactions with model-protein human serum albumin(HSA). HSA-adsorption data for PEI/ALG and CHT/ALG PEMs show the importance of theoutermost polyelectrolyte (PEL) layer for protein interactions: CHT- or PEI-terminated PEMs arehighly attractive to HSA, while ALG-terminated PEMs are repulsive, relevant for the generation ofprotein-active or protein-inert biomedical surfaces. The bound HSA is found to be located at the surfacerather than the inner region of the PEM.
Interfacial phenomena often play a key role in the research
and development of biomaterials, biomedical devices, phar-
maceutical formulations and separation. In that frame, the
interaction between proteins and polymer surfaces constitutes
an important phenomenon in colloid and materials science,[1–5]
where protein sorption is, on the one hand, desired for bioactive
applications (e.g., uptake of collagenized implants) and, on the
other, should be prevented for bioinert purposes (e.g., clotting
on medical devices, membrane fouling) in order to block or
delay further bioadhesion cascades.
Recently, much theoretical and experimental research, as
well as industrial development work, has been focused on
electrostatic forces between proteins and charged surfaces.
Experimentally, those charged surfaces can be provided by
single-polyelectrolyte or polyelectrolyte-brush layers,[6,7] self-
assembled monolayers (SAMs)[8,9] with charged end groups
and polyelectrolyte multilayers (PEMs).[10,11] This contribution
focuses on PEMs and their interactions with a model protein,
which has also been extensively studied by Voegel, Schaaf and
co-workers,[12–16] Salloum and Schlenoff,[17] Brynda at al.,[18]
Sukhishvili at al.[19] and Muller and co-workers.[20–22] Mean-
while it is commonly accepted, that PEM assemblies are
well-defined platforms for studying protein sorption due to
[*] Dr. M. Muller, B. Torger, B. KeßlerInstitute of Polymer Research Dresden e.V., Department ofPolyelectrolytes and Dispersions Hohe Straße 6, 01069Dresden, GermanyE-mail: mamuller@ipfdd.de
[**] This work is related to the Transregio SFB TRR 79 (M7) ofGerman Research Foundation (DFG) ‘‘Materials for tissue regen-eration in the systemically deseased bone’’ (University Giessen,University Heidelberg and Technical University Dresden).
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fundamental aspects, and for generating protein-inert or
binding surfaces for several applications like ophthalmic [23]
or bone implant related ones.[24] Some concerning questions are
still open. One of these questions is dedicated to the location of
the protein after its interaction with the PEM system. In that
context, certain reports claim migration into the PEM phase[17]
and evidence diffusion and embedding to a certain extent in the
PEM phase,[13] while other authors claim the interaction of
proteins to PEMs to be restricted to the surface.[25]
Herein, we would like to give a further contribution to that
issue. While in our former work this was already addressed
based on PEM systems that were composed of synthetic
polyelectrolytes (PELs),[20,22,25,26] herein we report PEM
systems that consisted of linear PEL components of natural
origin, claimed to be biocompatible. We chose two PEM
systems: poly(ethyleneimine)/alginate (PEI/ALG) and chit-
osan/alginate (CHT/ALG). In situ attenuated-total-reflection
Fourier transform infrared (ATR-FTIR) spectroscopy was
selected to address both the deposition and the protein
interactions related to these PEM systems. An
ATR-FTIR-spectroscopy attachment and measurement con-
cept that was introduced by Fringeli[27] was used, which we
have applied since the nineties on the characterization of PEM
deposition,[20] composition[21] and internal structure,[28] and
of the interactions of model polymers and especially PEM
films with proteins, peptides and drugs.[20,25,29–31]
Experimental
Chemicals and Solutions
Sodium alginate (ALG) (460 000 g mol�1) was purchased
from Kelco (San Diego, CA) and human serum albumin (HSA)
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Fig. 1. Principle of the in situ ATR-FTIR spectroscopy, featuring exponentiallydecaying evanescent waves at the optically dense/rare medium interface to probesurface-attached organic material. Reproduced with permission from ref.[24], 2007,Oldenburg Verlag.
(approximately 66 000 g mol�1, isoelectric point¼ 4.8) from
Sigma–Aldrich (Steinheim, Germany). Chitosan (CHT) (90.1%
degree of deacetylation, 300 000–400 000 g mol�1) was pur-
chased from Carl Roth GmbH (Karlsruhe, Germany). Acetic
acid was from Fluka (Steinheim, Germany). Poly(ethylenei-
mine) (PEI) (750 000 g mol�1) was from BASF-SE (Ludwig-
shafen, Germany). ALG solutions were prepared by dissol-
ving sodium alginate in Millipore water to a concentration of
0.005 M under stirring and CHT solutions were prepared by
dissolving the chitosan in a 0.002 M solution of acetic acid in
Millipore water. PEI solutions (pH�10) were prepared
without pH adjustment and used at a concentration of
cPEL¼ 0.005 M. HSA solutions were prepared by dissolving the
HSA in 1 mg ml�1 solutions of phosphate buffered saline
(PBS) (Sigma–Aldrich) to a concentration of 1 mg ml�1. All of
the PEL and protein solutions were prepared on the day of the
experiment and kept no longer.
PEM Preparation and Protein Adsorption
The PEMs were prepared by injecting the polycation (CHT
or PEI) solution, pure water, and the ALG solution in that
sequence into the in situ ATR cell (see below) as has been
described for other PEL systems.[29] An automated valve
system was used, by which the flow (ml min�1), the
adsorption time (min) and the rinsing time (min) could be
varied according to the following protocol: i) polycation
solution (adsorption), 5 ml min�1, 10 min; ii) Millipore water
(rinse) 5 ml min�1, 1 min; iii) ALG solution (adsorption),
5 ml min�1, 10 min; iv) Millipore water (rinse) 5 ml min�1,
1 min; v) continue at i).
For the protein-adsorption measurements, the HSA solu-
tions were injected into the in situ ATR cells manually using a
syringe at zero time.
ATR-FTIR Spectroscopy
For the infrared measurements, a commercial FTIR
spectrometer (IFS 55, BRUKER-Optics GmbH, Ettlingen,
Germany) equipped with a globar source and a mercur-
y-cadmium-telluride (MCT) detector was used. The PEM
growth and the protein adsorption were monitored using in
situ ATR-FTIR spectroscopy. The general set-up of the
ATR-IR-spectroscopy principle is given in Figure 1. The in
situ ATR cell (IPF Dresden) consisted of a silicon or
germanium trapezoidal internal-reflection element (IRE)
(50� 20� 2 mm2, KOMLAS GmbH, Berlin) housed by two
appropriate plates from both sides and sealed by O-rings,
resulting in four liquid compartments. The two upper ones
(front and back) sealed the sample (S) and the two lower ones
sealed the reference half (R) of one IRE. Single-channel
intensity spectra (IS, IR) were recorded from the S- and
R-halves of the ATR cell by shuttling them alternately in a
fixed IR beam that was provided by a special mirror
attachment (OPTISPEC, Zurich, Switzerland). The single-
beam sample-reference (SBSR) technique of Fringeli[31]
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resulted in proper spectral compensation, especially of the
solvent, and flat baselines, which is a prerequisite for
quantitative ATR-FTIR spectroscopy. In total, 200 scans in
packages of 50 scans were recorded at 2 cm�1 resolution for IS
and IR, respectively. The absorbance spectra, A, were
calculated by A¼ � log(IS/IR); no smoothing was applied.
Quantitative ATR-FTIR-Spectroscopy Analysis
Principally, quantitative ATR-FTIR-spectroscopy analysis
is based on a modified Lambert–Beer law:
AS ¼ N � " � c � dE;S (1)
Equation (1) includes the integrated absorbance of a given
IR band AS (cm�1), measured in s-polarization, the number of
active reflections N, the absorption coefficient e (cm mol�1),
and the film concentration c (mol cm�3). The effective
thickness dE,S (cm�1), due to Harrick [32], is given by Equation
(2) and is a function of the depth of penetration dP
(approximately 460 nm calculated from ref. [33, 34]), the
relative electrical-field component in the y-direction
EY ¼ 2cosuð Þ�
1�n231
� �½(approximately 1.53 in that work),
the refractive indices of the involved media n1 (Si, 3.5), n2
(PEM, protein, 1.5) and n3 (water, 1.33), the incident angle of
the IR light u (458) and the variable positions d1 and d2¼ d1þ d,
denoting the start and the end of the studied layer zone
(polymer, protein).
dE;S ¼n2 � dp � E2
Y
� �2n1 cosuð Þ exp � 2d1
dp
� ��exp � 2d2
dp
� �� �(2)
For polymer or protein layers starting directly at the
surface, d1¼ 0 and d2¼ d. For these systems, the absorbance AS
shows a dependence on dE,S as is given in Figure 2.[24]
This illustrates an approximate linear dependence of A on d
in the range d� 0–300 nm and an increasing insensitivity of the
ATR-FTIR-spectroscopy method to the outer polymer-film
regions with increasing thickness. Multiplying the film
concentration c by d, the surface concentration G (mol cm�2)
can be obtained. The time-dependent course A(t), for example
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0
0.2
0.4
0.6
0.8
1
0 2E-05 4E-05 6E-05 8E-05 1E-04
d / [cm]
A /
[cm
-1]
Fig. 2. Course of the ATR-measured absorbance AS, as a function of the variable layerthickness d (d1¼ 0) according to Equation (1 and 2), using scaled values for e andc. Reproduced with permission from ref. [24, 2007, Oldenburg Verlag.
Fig. 3. a) In situ ATR-FTIR spectra on the deposition of multilayers of CHT/ALG at aGe-IRE (pH¼ 3 (CHT) and pH¼ 7 (ALG)). ATR-FTIR spectra of PEM-1 to PEM-8are shown. Odd- (CHT-terminated) and even- (ALG-terminated) numbered PEMs areprinted bright and dark, respectively. b) Integrals of the diagnostic bands fromthe ATR-FTIR spectra in Figure 3a plotted versus adsorption step z. The course ofthe n(C�O) band integral was fitted by an exponential growth function of the typeA¼A0 � exp(a � z).
for protein adsorption, cannot be analyzed straight-forwardly,
since, besides G, the thicknesses d and dE,S are also time
dependent, due to Equation (3):
A tð Þ ¼N � " � G tð Þ dE;S d tð Þð Þ� �
½d tð Þ� (3)
However, for d< 300 nm, the ratio dE,S/d is approximately
constant and the absorbance A is roughly linear with the
surface concentration G. In that case, GProtein can be
determined assuming a box with a constant thickness
increment d¼ d2� d1, which the proteins fill by adsorption.
AFM Thickness Measurements
The thickness of the PEM films was determined using
atomic force microscopy (AFM) (Nanostation II, Bruker Nano,
Herzogenrath) applying scalpel-line cuts and measuring the
cut depth as described in ref. [33].
Results and Discussion
Herein we report in situ ATR-FTIR-spectroscopy studies on
PEMs consisting of the PEI or CHT polycation and the ALG
polyanion. Two phenomena of such biocompatible PEMs shall
be addressed, which are: 1) their growth or deposition and 2)
their interaction with proteins. The potential of ATR-FTIR
spectroscopy for the detection of interfacial sorption processes
is outlined: firstly, ATR-FTIR-spectroscopy data on the
deposition and thickness of PEMs of PEI/ALG and CHT/
ALG as a function of the adsorption step z are shown;
secondly, results on protein sorption for the two PEM systems
under both electrostatically attractive and repulsive condi-
tions are presented. For the electrostatically attractive
condition, results on the thickness dependence of the
adsorbed protein amount are presented, aimed at determining
the adsorption mechanism and the protein location.
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Deposition of Polycation/ALG Multilayers
CHT/ALG Multilayers
Typical in situ ATR-FTIR spectra for the sequential CHT/
ALG deposition process are shown in Figure 3a. From the
bottom to the top, the spectra are due to the actual
PEL-adsorbed amounts for the adsorption steps z¼ 1 to 8,
such that the first spectrum is due to the sorbed CHT amount
(‘‘PEM-1’’) after the Ge substrate had been firstly in contact
with the CHT solution, the second spectrum is due to the
sorbed CHT and ALG amount (PEM-2) after the pre-formed
CHT layer had been in contact with ALG solution, and so on for
PEM-3 to PEM-8. The ATR-FTIR spectra of the odd or
CHT-terminated PEMs are printed bright and those of the
even or ALG-terminated PEMs are printed dark. Qualitatively,
increasing IR signals are obtained in the ATR-FTIR spectra.
More quantitatively, in Figure 3b, typical deposition
profiles, which means the course of the integrals of diagnostic
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Fig. 4. a) In situ ATR-FTIR spectra of odd numbered PEM-1 to PEM-17 (from bottomto top) of PEI/ALG (pH¼ 10 (PEI), pH¼ 7 (ALG)) deposited onto Si-IRE in contactwith PBS solution (1 mg ml�1). b) Band integral of the n(COO�) band at 1593 cm�1
(scaling with ALG content) from the ATR-FTIR spectra on PEM-PEI/ALG (blackcubes) and thickness (AFM line-cut) versus adsorption step z (red points). The black andred full lines correspond to fits by an exponential function of the type A¼A0 � exp(a � z).
IR bands like the n(CH) of both the CHT and the ALG between
3000 and 2800 cm�1, the n(C¼O) at around 1720 cm�1 and the
n(COO�) at around 1590 cm�1 due to ALG in the protonated or
deprotonated form, respectively, and the n(C-O) at 1090 and
1030 cm�1 due to polysaccharide moieties of both the CHT and
the ALG are given. Overall, increasing and slightly modulated
courses (COOH, COO�) of these band integrals can be
identified. Concerning the overall increasing courses, the
polysaccharide-band integral, for example, was found to scale
exponentially with the adsorption step according to a function
of the type A¼A0 exp(a z). The respective fit is given in
Figure 3b. Exponential growth of PEMs has been observed for
various PEM types by Picart at al.[34] and Garza at al.[35] and
they explained this behaviour using a 3-zone model.
According to the 3-zone model, zone I is the initial zone of
the first few layers situated directly above the substrate and
zone III is the so called diffusion zone, in which, after the build
up of zone I, the PEM grows exponentially. In zone III, the
supplied PEL can ‘‘diffuse in and out’’ to compensate charges
at the surface by complexation. All of the PEL can participate
in complexation (the ‘‘Reservoir Effect’’) and the thickness
depends on the thickness increment. Zone II, between zones I
and III, is the so called ‘‘restructuration zone’’ in which the
PEL can no longer diffuse. It is formed when newly sorbed
PEL on top of zone III causes a release of PEL at the bottom of
zone III into zone II (at the II/III interface). When this final
process starts, the PEM is growing linearly with respect to the
adsorption step. Importantly, the PEM is in a non-equilibrium
state and all of these processes are time dependent. This
means that longer adsorption times enable the PEL to diffuse
in and out of the PEM for longer periods and to a higher
extent. Finally, this would favour linear growth. Up to now, no
general rules or predictions can be raised as to which systems
rather grow linearly and which exponentially. However,
Lavalle et al.[37] and Picart et al.[35] claim that PEMs of strong
PELs like poly(diallyldimethylammonium chloride)/poly(-
styrenesulfonate) were found to grow linearly and PEMs of
weak PEL were found to grow exponentially. Since the CHT/
ALG system is composed of weak PEL were found
exponential growth is in line with the observations of these
authors.
Concerning the modulated courses of the bands assigned
to the COOH and COO� moieties, the former go ‘‘up’’ in
the even ALG steps and ‘‘down’’ in the odd CHT steps and the
latter vice versa. This can be explained based on the exposure
of undissociated COOH groups in the ALG steps and their
deprotonation by complexation in the CHT steps, although
the pH of the CHT solution was 3. Another explanation
includes not only partial uptake, but also partial removal, of
PEL material whenever a new PEL solution is in contact with
the actual PEM. This supports the known growth behaviour of
PEMs; for example, as claimed by Kovacevic et al.,[38] that
there is a competition between PEL uptake to the PEM and
PEL removal from the PEM, resulting in a solution-complex
formation. A further explanation includes partial ‘‘diffusion
in’’ and ‘‘diffusion out’’ of PEL from the inner regions of the
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PEM to the outer regions upon exposure to the oppositely
charged PEL solution, which could be sensed by ATR-FTIR
spectroscopy and is in line with the accepted 3-zone model of
Picart, Lavalle and Garza and coauthors.[35–37] Additionally
the monotonously decreasing course of the integral of n(OH),
due to water, is shown, which reflects the increasing
displacement of water from close proximity to a larger
distance from the substrate surface.[20]
PEI/ALG Multilayers
Additionally, PEM films of PEI/ALG were deposited.
These PEM samples served as substrates of variable thickness
for the adsorption of the model-protein HSA, which will be
shown below. ATR-FTIR spectra of odd numbered PEM-1 to
PEM-17 of PEI/ALG are shown in Figure 4a.
The spectral intensity of the region between 1800 and
1300 cm�1 increased with the layer number z and, as
diagnostic bands for the deposition progress, the na(COO-)
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Fig. 5. a) In situ ATR-FTIR spectra for HSA adsorption (1 mg ml�1 in PBS buffer) atPEM-8 (lower panel) and PEM-9 (upper panel) of CHT/ALG recorded over 1 h. b)Adsorption kinetics of HSA at PEM-8 and at PEM-9 of CHT/ALG, respectively,according to Figure 5a.
band at around 1593 cm�1 and the ns(COO�) band at around
1410 cm�1 are of most significance. In Figure 4b, the na(COO�)
band is plotted versus adsorption step z. Since for PEM-17 we
obtained a thickness of d� 180 nm by the AFM line-cut
technique,[34] we are in the thin-film regime of ATR-FTIR
spectroscopy for the whole range of adsorption steps from
z¼ 1 to 17, meaning that the band integral of, for example,
na(COO-) is approximately linear with the thickness.[25,33]
Solely, for thicknesses d� 300 nm, this approximation is no
longer valid and the band-integral–thickness plots run into
saturation. Similar to CHT/ALG, the thickness of PEMs of
PEI/ALG only increased slightly from PEM-3 to PEM-7, but
started thereupon to grow significantly with z. As an
explanation, lateral fusing of individual deposition spots up
to z� 5–7, followed by the onset of vertical deposition for
z> 5–7, has been raised by several authors.[35,39] Similar to
CHT/ALG, the PEI/ALG deposition profile could be also
described by an exponential-model function, as shown in
Figure 4b. Since this system was also composed of a weak PEL,
the found exponential growth was not unexpected. This
exponential increase with adsorption step z was also
confirmed by thickness measurements using the AFM line-cut
technique on the same samples, as can be seen from the fit (red
line) of the experimental thicknesses (red points). This
observation is also important for analytical reasons, since it
validates experimentally the linearity between the AFM
thickness and the ATR-FTIR-spectroscopy adsorbed-amount
data, up to thicknesses of d< 300 nm, as is illustrated in
Figure 2.
Interaction of Polycation/ALG Multilayers with Proteins
Protein-adsorption studies were performed on these
polycation/ALG multilayers. For these studies, the PEM
films were rinsed with the buffer solution used for the
protein-adsorption experiment. We chose human serum
albumin (HSA) as a model protein for these studies. HSA
(1 mg ml�1) dissolved in PBS buffer (pH¼ 7.4, 1 mg ml�1)) has
a net negative charge, since the isoelectric point ranges at 4.8.
Influence of Outermost PEL Layer of PEM
Both the even ALG-terminated PEM-8 and the odd
CHT-terminated PEM-9 were contacted with the HSA
solution. The ATR-FTIR spectra, due to the difference between
the given PEM in contact with the PBS buffer and the given
PEM in contact with the HSA in the PBS buffer are shown in
Figure 5a. The lower set of spectra is for time-dependent HSA
adsorption at PEM-8 and the upper set is for that at PEM-9.
Qualitatively, the intensities of the protein-diagnostic amide-I
and amide-II bands were low for the HSA at PEM-8 and high
at PEM-9. Hence, we can conclude that HSA is strongly
adsorbed at the CHT-terminated PEM-9 due to electrostati-
cally attractive conditions and is repelled or weakly adsorbed
at the ALG-terminated PEM-8 due to repulsive conditions.
Similar effects were observed for HSA and other proteins at
PEMs of CHT/ALG (data not shown) and systems of
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commercial synthetic PELs like PEI/poly(acrylic acid).[20–22,29]
This confirmed that the strong dependence of protein
adsorption on the outermost layer charge is in line with the
findings of Ladam et al.,[14] especially for HSA at PEMs of
poly(allylamine)/poly(styrenesulfonate) (PAH/PSS) but also
for other proteins, and those of Van Tassel et al.[39] for the
interaction of fibronectin (FN) with the same PEM type (PAH/
PSS), which evidenced, similar to the work of Ladam et al., a
higher FN amount at the PAH-terminated PEM.
Furthermore, from Figure 5b, for the HSA interaction with
PEM-9 of CHT/ALG, a significant time dependence was
observed, which was not found for HSA at PEM-8. This can be
explained by two scenarios: (A and B):on the one hand,
strongly bound HSA at PEM-9 is adsorbed in quantities far
larger than for a monolayer by long-range electrostatic forces
or by partial protein surface aggregation due to short-range
forces. On the other hand, HSA at PEM-9 might be able to
diffuse into the PEM phase. ATR-FTIR spectroscopy is
sensitive to both scenarios, since, in the first case, the protein
is increasingly accumulated at the outside of the PEM having
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an approximate thickness of 100 nm, so that the bound protein
is sensed by a still-high evanescent-field intensity, leading to
an increase of the amide-II integral. In the second case, the
protein increasingly moves to positions where the evanescent-
field intensity is increasingly higher, leading to a time-
dependent increase of the amide-II integral as well.
Influence of PEM Thickness
To get at least a qualitative, hand-waving argument for one
or the other scenario, we measured the adsorbed-HSA sorbed
amounts at PEM-z of PEI/ALG, which were deposited
applying different adsorption steps z. In other words, the
HSA sorption was measured at PEMs with different
adsorbed-amount levels or thicknesses. The ATR-FTIR spectra
for HSA sorption at PEM-7 (bottom set), PEM-9 (middle set)
and PEM-15 (top set) of PEI/ALG are given in Figure 6a.
Spectra recorded after 5 to 60 min are plotted on the same
baseline for each of the sets. Intense amide-I and amide-II
Fig. 6. a) ATR-FTIR spectra for the sorption of HSA (1 mg ml�1) at PEM-7, PEM-9and PEM-15 of PEI/ALG. The spectra recorded over 1 h are shown. b) Amide-II integralscaling with sorbed HSA amount versus n(COO-)-band integral scaling with PEM-PEI/ALG amount (ALG content) (black cubes). These data were fitted by the functiongiven in Equation 4 (full line).
ADVANCED ENGINEERING MATERIALS 2010, 12, No. 12 � 2010 WILEY-VCH Verl
bands at 1654 and 1547 cm�1 were obtained, which are dia-
gnostic for protein binding or accumulation at the PEM/water
interface. According to classical amide-band-assignment
papers,[40–43] these band positions indicate further that HSA
is an a-helix-rich protein. No large variations of the band
intensities were observed as a function of time in each of the
three sets, which suggests rapid sorption processes and
adsorption saturation after around an hour. Qualitatively, the
intensity of the amide-II band, which is related to the adsorbed
amount of HSA, increased from PEM-7 to PEM-9 and then
dropped again for PEM-15.
More quantitatively, in Figure 6b, the amide-II-band
integrals, representing the protein-bound amount, are plotted
versus the n(COO�) band integrals of various PEM-z of PEI/
ALG, representing the PEM-deposited amount or thickness.
Interestingly, after the initial rise from PEM-7 (first data point)
to PEM-9, a further, continuous decrease of the amide-II-band
integral was observed from PEM-9 to PEM-17. While the
initial rise can be explained by the completion of lateral
homogeneity of the PEM, the drop from PEM-9 to PEM-15 can
be explained in the light of earlier findings on HSA adsorption
at PEI/PAC multilayers.[24] Therein, a maximum of the
amide-II-band integral was reached at PEM-5, and for PEM-7,
PEM-9 and PEM-11 it subsequently decreased. We concluded,
that the protein was preferentially bound on top of the PEM
films for all of the PEM samples (scenario A), since the
evanescent wave senses the protein layer less the higher the
PEM film thickness.
Scenario B includes migration of the protein into the PEM
phase, which would qualitatively imply that the amide-II
signal should not decrease upon an increase in thickness. The
following, related functional relationship was introduced in
an earlier paper,[24] and describes the exponentially decaying
sensitivity of the ATR-FTIR-spectroscopy experiment for HSA
layers on top of PEM films with the PEM-deposited amount or
thickness (scenario A):
AAmide�II¼K exp �L �AnðCOO�Þ� �
�exp �L � An COO�ð ÞþA�n COO�ð Þ
� � h i(4)
In Equation (4), K is a constant, An COO�ð Þ is, in principle,
approximately linear with d1 (the thickness of the PEM
without any protein) and A�n COO�ð Þ is approximately linear
with d2� d1 (the thickness of the protein layer), which was
assumed to be constant for PEM-7 to PEM-17. L corresponds to
a damping parameter, which is further related to dP. This
function was used to fit the experimental data and is shown as
the full black line in Figure 6b, whereby the parameters K, L
and A�n COO�ð Þ were optimized. The data set included various
An COO�ð Þ values, according to various PEM-z, and their
corresponding AAmide-II values (protein). Obviously, the
experimental data could be fitted sufficiently by that simple
approach, which favours scenario A and disfavours scenario B
for the PEMs of PEI/ALG. A related scheme is given in
Figure 7, illustrating that scenario A has to be preferred to
describe our experimental data. We are aware that these
ag GmbH & Co. KGaA, Weinheim http://www.aem-journal.com B681
RESEARCH
ARTIC
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M. Muller et al./Deposition of Polycation/ALG Multilayers
Fig. 7. ATR-FTIR-spectroscopy detection of protein (bright grey) adsorption at PEM-z of PEI/ALG (grey) deposited on IRE (shaded) as a function of adsorption step z, consideringthe exponentially damped evanescent IR field (dark grey). Scenario A in Figure 7A implies protein sorption limited to the PEM surface region. Scenario B in Figure 7B implies proteinsorption within the whole PEM phase.
results, found for HSA at PEI/ALG, are conflicting with other
results on PEM/protein interactions. In that context, Salloum
and Schlenoff[17] reported migration into the PEM phase and
Szyk at al.[13] evidenced diffusion and embedding to a certain
extent in the PEM phase, while in former reports of our group,
other systems like PEI/PAC also showed that protein
interactions were restricted to the surface[24] and that the
outermost layer charge and the net charge of the protein
determined by its isoelectric point (IEP) was crucial for the
bound protein amount,[20–22,25,29] which is principally in line
with other reports of Ladam et al.[14] and Van Tassel et al.[39]
Conclusion
In situ ATR-FTIR-spectroscopy studies on the deposition of
biocompatible PEMs consisting of chitosan (CHT) and sodium
alginate (ALG), and of poly(ethyleneimine) (PEI) and alginate,
and their interaction with model-protein human serum
albumin was studied. Firstly, the ATR-FTIR-spectroscopy
data showed an exponential PEM film growth and a
modulated course of the composition as a function of the
adsorption step z for both the PEI/ALG and CHT/ALG PEM
systems. The modulated composition was interpreted by
protonation-degree changes, as well as by competition
between PEL uptake and PEL rupture, when a given PEL
was in contact with the appropriate PEM. Secondly, the
ATR-FTIR-spectroscopy data showed the importance of the
type and charge of the last adsorbed layer, when model
proteins like human serum albumin (HSA) interact with the
PEM. CHT- or PEI-terminated PEMs were attractive to HSA,
while ALG-terminated ones were repulsive, which suggests
the ability of creating protein-active or protein-inert biome-
dical materials by this simple surface-modification technique.
Thirdly, ATR-FTIR-spectroscopy data on HSA adsorption at
odd-numbered, oppositely charged PEM surfaces as a
function of PEM thickness evidenced that the bound proteins
are located at the surface region rather than in the interior of
the PEM.
B682 http://www.aem-journal.com � 2010 WILEY-VCH Verlag GmbH & C
Received: June 16, 2010
Final Version: August 27, 2010
Published online: November 17, 2010
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