in situ atr-ftir spectroscopy on the deposition and protein interaction of polycation/alginate...

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DOI: 10.1002/adem.201080059
In 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: [email protected]

[**] 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).

B676 wileyonlinelibrary.com � 2010 WILEY-VCH Verlag GmbH & Co.

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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M. Muller et al./Deposition of Polycation/ALG Multilayers

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]

ADVANCED ENGINEERING MATERIALS 2010, 12, No. 12 � 2010 WILEY-VCH Verl

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

ag GmbH & Co. KGaA, Weinheim http://www.aem-journal.com B677

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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.

B678 http://www.aem-journal.com � 2010 WILEY-VCH Verlag GmbH & C

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


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


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).


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


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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.


Received: June 16, 2010

Final Version: August 27, 2010

Published online: November 17, 2010

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