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This is a Post-print of a manuscript, which appeared in:

Thin Solid Films

Please cite this publication as follows:

M. Müller, B. Torger, E. Bittrich, E. Kaul, L. Ionov, P. Uhlmann, M. Stamm,

In-situ ATR-FTIR for characterization of thin biorelated polymer films,

Thin Solid Films 2014, 556, 1-8.

You can download the published version at:

http://dx.doi.org/10.1016/j.tsf.2013.12.025

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In-situ ATR-FTIR for characterization of thin biorelated polymer films

M. Müllera b

, B. Torger a, b

, E. Bittricha, E. Kaul

a,b, L. Ionov

a ,b, P. Uhlmann

a, M. Stamm

a, b

aLeibniz Institute of Polymer Research Dresden (IPF Dresden), Hohe Straße 6, 01069

Dresden, Germany

bTechnical University of Dresden (TUD), Department of Chemistry and Food Chemistry,

01062 Dresden, Germany

E-mail: mamuller@ipfdd.de

Abstract

We present and review in-situ-attenuated total reflection Fourier transform infrared (ATR-

FTIR) spectroscopic data from thin biorelated polymer films useful for the modification and

functionalization of polymer and inorganic materials and discuss their applications related to

life sciences. A special ATR mirror attachment operated by the single-beam-sample-reference

(SBSR) concept and housing a homebuilt thermostatable flow cell was used, which allows for

appropriate background compensation and signal to noise ratio.

ATR-FTIR data on the reactive deposition of dopamine on inorganic model surfaces are

shown. Information on the structure and deposition pathway for such bioinspired melanin-like

films is provided. ATR-FTIR data on thermosensitive polymer brushes of poly(N-

isopropylacrylamide) (PNIPAAM) is then presented. The thermotropic hydration and

hydrogen bonding behaviour of PNIPAAM brush films is described. Finally, ATR-FTIR data

on biorelated polyelectrolyte multilayers (PEM) are given together with details on PEM

growth and detection. Applications of these latter films for biopassivation/activation and local

drug delivery are addressed.

Keywords

ATR-FTIR, SFM, polymer film, polydopamine, poly(N-isopropylacrylamide) brush,

polyelectrolyte multilayer, protein adsorption, drug release

Introduction

Surface modification using thin biorelated polymer films is an important issue for the

research, development and production of biomedical materials and devices including bone

implants, contact lenses, blood contacting stents, and catheters. Polymer coatings are aimed at

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biopassivation such as the prevention of unspecific protein adsorption or cell adhesion as was

shown by polyethylene glycol modified surfaces [1]

and bioactivation like the exposure of

targeting molecules at the surface [2]

or the inclusion of elutable drugs.[3]

The characterization

of both deposition and function of these coatings at the model, in-vitro, and in-vivo levels are

important tasks for their future application in biomedicine.

Here, we present and review in-situ ATR-FTIR data on three examples of thin biorelated

polymer coatings on model substrates such as silicon and germanium. These are

polydopamine, poly(N-isopropylacrylamide), and polyelectrolyte multilayers of

poly(etyhleneimine) and sodium alginate, which are all usable for the modification and

functionalization of biomedical polymer as well as inorganic materials in applications related

to life sciences. A dedicated ATR mirror attachment (OPTISPEC, Zürich) housing a

homebuilt transparent and thermostatable flow cell is operated by the single-beam-sample-

reference (SBSR) concept,[4]

which allows for appropriate background compensation and

signal/noise ratio.

Experimental details

1.1. Polydopamine films

Polydopamine (PDA) films were prepared by injecting solutions of 2 mg dopamine (Sigma)

in 1 ml 0.001M Tris(hydroxymethyl)-aminomethan (TRIS) buffer (pH = 8.5) into the sample

(S) compartment of the in-situ-ATR-FTIR cell (see below) housing a Ge internal reflection

element (IRE) and allowed to react as described in reference.[5]

1.2. PNIPAAM brush films

Poly(N-isopropylacrylamide) (PNIPAAM) brush films were prepared by two approaches.

According to the grafting-to-approach described in reference [6] Si internal reflection

elements (IRE, see below) were spincoated by a 0.01% (w/w) poly(glycidylmethacrylate)

(PGMA) solution in tetrahydrofuran (THF) and dried for 20 min in a vacuum oven at 100°C.

Onto this PGMA precursor, a 1% (w/w) solution of carboxylic acid terminated PNIPAAM

(50 kg/mol) was spin coated and surface reacted by drying at 150°C in a vacuum oven.

According to the grafting-from-approach on silicon wafers described in reference [7] the

surface of the Si IREs was treated by alkaline hydrogen peroxide solution

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(NH4OH/H2O2/H2O-1:1:1), thoroughly rinsed and dried. Thereafter, the IRE surface was

reacted with a 2% (v/v) ethanol solution of 3-aminopropyltriethoxysilane (APTES) (2h, RT).

The modification to the initiator was achieved using 0.5% (v/v) -bromo-isobutyrylbromide

and 1 % (v/v) triethylamine in dichloromethane (2h, RT) according to Matyjaszewski.[8]

PNIPAAM brushes were grafted via a modified ATRP using an ethanol solution of NIPAAM

(1g/1ml) and CuBr2/PMDTA as a catalyst and ascorbic acid as a reducing agent (2 h, RT).

1.3. Polyelectrolyte multilayer films

Polyelectrolyte multilayer (PEM) films were prepared by consecutively injecting

poly(ethyleneimine) (PEI) solution, pure water and sodium alginate (ALG) or poly(acrylic

acid) (PAC) solution, into the in-situ-ATR cell (see below) as described for other PEL

systems in reference [9]. We used the polyelectrolytes (PEL) branched-PEI (750kg/mol,

BASF, Germany), linear-ALG (460kg/mol, Kelco, USA), and linear-PAC (50kg/mol,

Polysciences, USA) based on 0.005M PEL solutions.

1.4. Proteins, drugs

Human serum albumin (HSA, 66kg/mol, Sigma, Germany) was the standard protein and

streptomycin (STRP, Carl Roth, Germany) the standard drug in these experiments. HSA was

applied as 1 mg/ml solutions in phosphate buffered saline (PBS) and STRP was applied at the

concentration of 0.001M solutions in the unbuffered original 0.005M PEL (PEI, ALG, PAC)

solution.

1.5. in-situ ATR-FTIR spectroscopy

In Fig. 1. the in-situ ATR-FTIR concept, and in Fig. 2 the transparent thermostatable flow cell

(M.M. IPF Dresden) with separate sample and reference compartments around Si, Ge or ZnSe

internal reflection element are shown.

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Fig. 1. Scheme of the in-situ ATR-FTIR setup

including polarized light.

Fig. 2. Transparent in-situ ATR-FTIR cell

housing the Ge (Si, ZnSe) IRE used in the

deposition experiments. B: Back cell

component, F: Front cell component, S:

Upper sample (liquid) compartment, R:

Lower reference (liquid) compartment

(from [5], Supporting Information).

The flow cell is situated in an ATR mirror attachment (OPTISPEC, Zürich), which is

integrated into an FTIR spectrometer (Bruker Optics, Ettlingen, Germany). The flow cell is

shuttled between two different heights with respect to the fixed IR beam according to the

single-beam-sample-reference (SBSR) concept,[4]

which enables the quasi-simultaneous

measurement of sample (S) and reference (R) intensity spectra. Processing these S- and R-

intensity spectra (A = log IS/IR) provides in-situ ATR-FTIR spectra featuring flat baselines by

appropriate background compensation with a convenient signal/noise ratio. These in-situ

ATR-FTIR spectra can be quantitatively evaluated.

1.5.1. Surface concentration

From band integrals A of in-situ ATR-FTIR spectra, the surface concentration = c d (c:

concentration, d: geometric thickness) of IR-active functional groups in polymer films can be

calculated based on the formalisms of Harrick[10]

and Fringeli[4]

using eqns. 1-3. For this

calculation, the number of reflections N, absorption coefficient , the effective thickness dE

which is a function of refractive index ratio n21 = n2/n1, depth of penetration dP, electric field

amplitude E with its relative electric field components Ex, Ey, Ez, the incident angle , and d

must be known.

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A = N c dE (1)

dE = n21dPE2/(2 cos ) [1-exp(-2d/dP)] (2)

= c d (MIN = 0.1 nM/cm2) (3)

Strictly, the equations hold only for band integrals measured by parallel (A║) or vertical (A)

polarized IR light. However, for randomly-oriented polymer samples, there is an

approximately constant factor F between, for example, A and band integrals measured by

unpolarized light (A). Therefore, for the calculation of eq. 3), it can be empirically

approximated that A = F A and E = Ey (Ex, Ey, Ez values are known from Harrick [10]

).

Scanning force microscopy (SFM)

SFM images were measured from polymer films using Nanostation II of Bruker Nano GmbH

(Karlsruhe, Germany). Silicon probe tips from Nanosensors (Darmstadt, Germany) with apex

radii of around 10 nm were used. Images were measured in topography, error and phase mode

and scanning parameters were optimized by minimizing the error mode signal. Images and

surface profiles were generated from SFM raw data by the SISCANPro software (Bruker

Nano GmbH, Karlsruhe, Germany). Thicknesses of polymer films were determined by

imaging the surface profile around careful scalpel cuts into the polymer film not damaging the

substrate. The distance between the bottom of the cut and the undamaged film surface was

taken as film thickness.

Results and discussion

In the following sections, results on the characterization (1.) and application (2.) of thin

biorelated polymer coatings based on in-situ ATR-FTIR spectroscopy, are presented and

reviewed.

1. In-situ ATR-FTIR characterization of biorelated polymer films

1.1. Reactive dopamine deposition

Mussels adhere to surfaces via secreted byssus threads, which are composed of proteins

containing a large amount of amino acid L-dihydroxyphenylalanin (L-DOPA). L-DOPA is

able to form reactive crosslinking biopolymerisates. Recently it was found by Messersmith,[11]

that dopamine (DA, Fig. 1B), which is the structural analogue of L-DOPA, also forms thin

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crosslinked polymerisate films with thicknesses d 50 nm simply by adsorption from alkaline

solutions onto various material surfaces. Such bioinspired polydopamine (PDA) films are

appealing for biomedical, cosmetic (UV protection) and even electronic applications.[11]

The

chemistry is claimed to be known on the monomer level, where after a sequence of pH-

dependent oxidation steps, DA is transformed into the reactive monomer indole-5,6-quinone

(IQ)[12]

as shown in Fig. 3a. However, neither the subsequent surface-mediated

polymerisation mechanism, nor the exact structure of PDA is fully resolved. Actually, the

structure of PDA is claimed to be similar to the skin pigment melanin.[12]

We studied DA deposition by in-situ ATR-FTIR spectroscopy as a function of time under a

variety of parameters: pH, concentration, ionic strength, and substrate type.[5]

In Fig. 3b ATR-

FTIR spectral data concerning the pH dependence of DA deposition on Ge model substrates

are shown. To our knowledge this is the first in-situ-ATR-FTIR spectral data. At pH = 8.5,

ATR-FTIR spectra with increasing signal intensities at 1490, 1435 and 1250 cm-1

(polyphenoles) were obtained, while for pH = 6.2, no significant peaks were recorded.

However, ATR-FTIR spectra for the former case differ very much from that of synthetic

melanin (data not shown).

Fig. 3a. Proposed

reaction mechanism at

the monomer level.

Fig. 3b. In-situ ATR-FTIR spectra for the time-dependent deposition

of PDA layers on Ge model substrates for pH = 8.5 and pH = 6.2

(spectra correspond to times from bottom to top: 5, 10, 15, 20, 30,

60, 120, 180, 1200 min) (from [5]

with kind permission of ACS)

NH2

OH

OH

HN

O

O

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In Fig. 3c, the integrated areas under the peaks are plotted versus time. The kinetic DA

deposition data (A(t)) can be represented by a simple exponentially damped function of the

type A(t) = A0 (1 - exp(-k1 t)), as is also shown in Fig 3c.

Fig. 3c. Plot of the IR band integrals at 1490, 1435 und 1250 cm-1

versus time and fits based

on the Langmuir adsorption model. (from [5]

with kind permission of ACS)

A kinetic rate constant of k1 0.013 min-1

was determined, which qualifies as a rather slow

process compared to protein adsorption (k1 0.08 min-1

, unpublished data). Furthermore, the

concentration (c) dependence of the DA deposition can be fit by a simple Langmuir

adsorption model [5] to A(c) = A0/(a + c) (see Fig. 4a). Scanning force microscopy (SFM)

images of the PDA film suggest granular individual particles[5]

as shown in Fig. 4b. Thus,

preformed PDA particles in solution appear to be the film-forming species. Such preformed

PDA particles are obvious by visual inspection of the reactive DA solution, which becomes

blackish with time and by dynamic light scattering studies revealing particle sizes around 50

nm. Since PDA film thicknesses in this range were reported [11]

and also measured by us using

SFM[5]

, we suggest monolayers of preformed reactive PDA particles as the film architecture.

PDA coatings will be considered for use in drug delivery applications.

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Fig. 4a. Adsorption isotherms for DA

deposition based on plots of ATR-FTIR

peaks at 1490, 1435 and 1250 cm-1

versus the

DA concentration. (from [5]

with kind

permission of ACS)

Fig. 4b. SFM image (phase, 2 x 2 m2) of

PDA film deposited onto Ge IRE substrates.

(from [5]

with kind permission of ACS)

1.2. Swelling of ultrathin PNIPAAM brush films

In the second example, results for thermosensitive polymer brushes based on poly(N-

isopropylacrylamide) (PNIPAAM) chemically attached to silicon substrates are presented.

Chemical attachment was achieved by both the grafting-to[6]

and the grafting-from[7]

approach

(see Experimental Details). In Fig. 5a, ATR-FTIR spectra of a 0.05M PNIPAAM solution and

of a PNIPAAM brush film (grafting-to) on silicon in the dry state are shown. Given a

thickness of around 10 nm for the brush film based upon ellipsometry data, a well-resolved

ATR-FTIR spectrum with appropriate baseline and signal/noise ratio was obtained. However,

the ATR-FTIR spectrum of a 0.05M PNIPAAM solution did not reveal such high spectral

quality. This illustrates the general sensitivity of the ATR method for macromolecules located

directly on optical denser media, where the interacting electric field amplitude of the

evanescent wave is maximum. In addition, the concentration of PNIPAAM molecules in a

0.05 M solution is less than the concentration of PNIPAAm molecules in the brush.

Otherwise, the ATR-FTIR spectrum in the solution should be more intense than that of the

brush due to the higher refractive index of water (n3 = 1.33) compared to air (n3 = 1).

Nevertheless, from the ATR-FTIR spectrum of the 0.05 M PNIPAAM solution, the

0.0

0.5

1.0

1.5

0 0.005 0.01 0.015 0.02

Ban

d i

nte

gra

l / [c

m-1

]

Concentration / [Mol/L]

1250/TRIS1490/TRIS1435/TRISFIT

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10

absorption coefficient of the Amide II band (Amide I interferes with the (OH) of bound

water) can be determined using eq. 1. Based on this value of = 7.13 x 107 cm/mol for the

Amide II band, a surface concentration of = 0.90 g/cm2 was determined, which is close to

the value of = 1.08 g/cm2 obtained by ellipsometry.

[6] In Fig. 5b, in-situ ATR-FTIR

spectra from ultrathin PNIPAAM brush films in contact with a PBS buffer are shown at

temperatures T = 22°C - 44°C.[13]

Fig. 5a. In-situ-ATR-FTIR spectra from

0.05M PNIPAAM solution (bottom) and on a

PNIPAAM (grafted-to) brush film on silicon

in the dry state (top).

Fig. 5b. In-situ-ATR-FTIR spectra from

amide bands centered at around 1631 and

1555 cm-1

of PNIPAAM (132kg/mol,

grafted-to) brush film in contact with PBS

recorded at temperatures T = 22 (bottom),

26, 28, 30, 32, 34, 35, 38 to 44°C (top).

(from [13]

with kind permission of ACS).

Fig. 5b shows that the Amide I band shifts up in wavenumber with temperature and the

Amide II band shifts down. Hence, PNIPAAM brushes show similar thermotropic behaviour

as PNIPAAM solutions. The wavenumber positions of the peak maxima are plotted versus

temperature in Fig. 6a and the respective integrated peak areas of the Amide I, Amide II and

additionally the a(CH3) band of the isopropyl group (spectral data not shown) are shown

versus temperature in Fig. 6b. Amide I and II band positions of PNIPAAM are diagnostic of

the type and degree of hydrogen bonding, which is described for PNIPAAM solutions in

references [14]

and [15]

. For temperatures T < LCST (lower critical solution temperature), where

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PNIPAAM is claimed to be in the coiled (soluble) state, the Amide I band position is lower

and the Amide II band position higher than for T > LCST, where PNIPAAM is claimed to be

in a globular (less soluble) state. Lower Amide I band positions are due to hydrogen bonding

between the amide C=O and water (C=O …

H …

O-H), weakening the C=O double bond

character and decreasing its force constant. Higher Amide II band positions are due to

hydrogen bonding between amide N-H and water (N-H …

O-H2) restricting the (NH)

bending mode and increasing its force constant. Obviously, Amide I band positions shift

continuously with increasing T over the entire T range (Fig. 6a, top), while Amide I and II

intensities were rather invariant from T = 22 - 32°C, but step-like increases were found for T

> 32°C (Fig. 6a, bottom). These step-like intensity changes of Amide I and Amide II bands

are due to a sudden increase of the PNIPAAM segment concentration in the brush for T >

LCST, in contrast to the rather continuous hydrogen bonding changes. Therefore, we suggest

that a step-like coil-to-globule transition of PNIPAAM brushes takes place when the

continuous hydrogen bonding changes have reached a certain critical point at T = LCST.

Fig. 6a. Wavenumber positions of the Amide I

and II bands from the ATR-FTIR spectra in Fig.

5b plotted as a function of T. (from [13]

with

kind permission of ACS).

Fig. 6b. Integrated peak intensities of

Amide I, Amide II and a(CH3) bands

from the ATR-FTIR spectra given in Fig.

5b as a function of T. (from [13]

with kind

permission of ACS).

Alternatively, Amide I and Amide II band shifts can be analyzed by lineshape analysis (LSA).

Fig. 7 compares LSA results for spectra obtained from PNIPAAM solutions by Maeda [15]

(Fig. 7a) and our own LSA results on ATR-FTIR spectra of PNIPAAM brush films (Fig. 7b).

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Fig. 7a. Results of line shape analysis on

FTIR spectra of PNIPAAM solution in D2O

at T < LCST (“Tp”) and T > LCST (“Tp”).

Solid lines correspond to original spectral

data and fitted components, dashed ones to

simulated ones. (from [15]

with kind

permission of Wiley)

Fig. 7b. Results of line shape analysis on

ATR-FTIR spectra of PNIPAAM brush

(grafted-to) films at T < LCST (22°C, top)

and T > LCST (40°C, bottom) in contact

with H2O. Original spectra are indicated by

solid lines, fitted components and simulated

spectra by dashed lines.

The peak centered at 1625/1627 cm-1

is assigned to external (segment/water) hydrogen

bonding, while the peak centered at 1650/1652 cm-1

is due to internal segment/segment

hydrogen bonding. For the spectrum of PNIPAAM in D2O solution at T < LCST, only one

Amide I peak at around 1625 cm-1

is required to fit the data, while for T > LCST two peaks

centered at 1650, 1625 cm-1

(D2O) are required. In contrast, in ATR-FTIR spectra from

PNIPAAM brush films in H2O, two peaks at 1652 and 1627 cm-1

are required for T both

below and above LCST. This shows clearly, that for T < LCST in the solution state,

PNIPAAM is fully dissolved and in a coiled conformation, while for T > LCST PNIPAAM is

less soluble and in a globular state as expected. However, since for the brush-state films two

components are required for both T < LCST and T > LCST, we conclude that in the brush

state the attached PNIPAAM chains are never in a fully dissolved or coiled state, but always

contain globular portions, a larger concentration at T > LCST and a lower at T < LCST.

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1.3.Layer-by-layer deposition of polyelectrolyte complex films

In this section, we review in-situ ATR-FTIR data on polyelectrolyte multilayers (PEM),

which are deposited by the layer-by-layer (LbL) technique reported by Decher.[16]

ATR-FTIR

spectroscopy of PEM films provides information concerning deposition mechanism,[17]

composition,[18]

and orientation.[19]

Here, ATR-FTIR data on LbL deposited

poly(ethyleneimine)/alginate (PEI/ALG) multilayers on silicon substrates [20]

are reviewed.

Fig. 8a. shows in-situ ATR-FTIR spectra over the range 1800-1300 cm-1

for PEI/ALG

multilayers for odd layer numbers: 1, 3, 5 … 17.[20]

Fig. 8a. in-situ ATR-FTIR spectra of PEM-1

to PEM-17 of PEI/ALG multilayers

deposited from 0.005 M PEL solutions on

silicon in contact with water (rinsing). (from [20]

with kind permission of Wiley)

Fig. 8b. aCOO-) integrated peak intensities

(ALG) from in-situ ATR-FTIR spectra of

Fig. 8a and corresponding thicknesses (SFM,

dry state) of PEM of PEI/ALG as a function

of adsorption step z. (from [20]

with kind

permission of Wiley)

Thus, spectra were obtained from the first adsorbed PEI layer (“PEM-1”); the next adsorbed

PEI/ALG/PEI layer (PEM-3) in the multilayer stack, up to PEM-17 were acquired and

compared. The a(COO-) band at 1593 cm

-1 and s(COO

-) band at 1410 cm

-1 of ALG (see Fig.

8a) were used as diagnostic markers for the PEM deposition process. In Fig. 8b, the

respective integrals of the a(COO-) band are plotted versus the adsorption step z = 1, 3, 5 …

17 (cube symbols). ATR-FTIR spectroscopy is characterized by an exponential decay of

sensitivity for processes taken place at larger distances from the ATR crystal surface. Thus,

for film growth processes, there is a certain film thickness at which the evanescent wave is no

0

50

100

150

200

0

2

4

6

8

10

0 2 4 6 8 10 12 14 16 18

Th

ick

ne

ss

/ [

nm

]

(C

OO

- )-I

nte

gra

l / [c

m-1

]

Adsorption step z

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longer sensitive to further deposition. For large thicknesses, the integrated peak intensity of,

for example, a(COO-) versus the adsorption step reaches a plateau following a damped

exponential curve (1 – exp(-d/dP)), where d and dP are the geometric thickness and the depth

of penetration. However, for small thicknesses d < 200 nm, ATR-FTIR spectroscopy senses

film growth in a linear fashion such that the measured band integral A is approximately

proportional to d. In Fig. 8b, an exponential increase is observed for the a(COO-) band. That

is, the polysaccharide band integral was found to scale exponentially with the adsorption step

according to a function of type A = A0 exp(a z), in which a is a growth parameter and z is the

adsorption step number. The fit is also shown in Fig. 8b (black line).

The geometric film thickness of PEI/ALG-17 was measured by SFM (see Experimental

Details). In Fig. 8b these thicknesses of PEM-PEI/ALG (dry state) are also plotted as round

red symbols versus z. An exponential increase of d with increasing z and, for PEM-17,

d = 180 nm was obtained. Thus, our ATR-FTIR data varied approximately linearly with the

SFM data and therefore the exponential growth found by ATR-FTIR is confirmed.

Exponential growth of PEM has been observed for various PEM types by Picart[21]

and

Garza[22]

and they explained this behaviour based on high PEL diffusivity and a 3-zone-

model, especially when weakly-charged PEL or higher salinity was used. According to a

recent compilation of this model,[20]

zone I is the initial zone of the first few layers situated

directly above the substrate and zone III is the diffusion zone, in which after build up of

zone I, PEM grows exponentially. In zone III, the supplied PELs can “diffuse in and out” to

compensate charges at the surface by complexation. All PEL can participate in complexation

(“Reservoir Effect”) and thickness depends on the thickness increment. Zone II between I and

III is the so called “restructuration zone” in which PEL can no longer diffuse. It is formed

when new sorbed PELs on top of zone III cause a release of PELs at the bottom of zone III

into zone II (at the interface II/III).[20]

2. Applications of selected polymer films

Application examples of the functional coatings analyzed in Section 1 are considered for

binding/repulsion of proteins (2.1) and the controlled release of drugs (2.2). In addition, the

potential of in-situ-ATR-FTIR spectroscopy as a screening tool to optimize protein interaction

or drug formulation is discussed.

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2.1. Protein interaction of polymer coatings

All polymer coatings described in Section 1 have potential for either bioinert or bioactive

functionalization of biomedical materials or surfaces. Using ATR-FTIR spectroscopy results

concerning protein composition and bound amount,[9]

location of the adsorbed proteins[23]

and

secondary structure[24]

are possible based on the integrated areas of the diagnostic Amide I

and Amide II bands. Example results are presented for the adsorption of the model protein

human serum albumin (HSA, isoelectrical point IEP = 4.7).

In Fig. 9a, HSA adsorption on PDA films is shown in comparison to adsorption on the bare

Ge substrate. The maximum Amide II band integral recorded for HSA adsorption on Ge was

taken as 100%. Significantly, HSA adsorption on PDA decreased by approximately 50%.

Fig. 9a. Relative adsorbed

amounts of HSA versus time

at bare Ge and at PDA film.

Fig. 9b. Relative adsorbed

amounts of HSA versus time

at bare Si and at PNIPAAM

(grafted-from) brush films for

T = 25°C and T = 40°C.

Fig. 9c. Relative adsorbed

amounts of HSA versus time

at PEM-4 and PEM-5 of

PEI/PAC and at PEM-8 and

PEM-9 of PEI/ALG.

HSA adsorption on PNIPAAM brush films was compared to that on Si substrates at both

temperatures T of 25 °C and 40 °C. As shown in Fig. 9b, PNIPAAM brush films exhibit

remarkably reduced HSA adsorption compared to bare Si substrate at both temperatures. In

these series on HSA adsorption, PNIPAAM brush films prepared by the grafting-from

concept (see Experimental Details) were used, which showed similar thermotropic behaviour

compared to those prepared by the grafting-to concept. While a slight effect of temperature

was observed for bare Si (higher HSA adsorption at T = 40°C), no such effect was seen on

PNIPAAM films. This effect was interpreted in terms of the strongly bound water on

PNIPAAM films, which prevents further attractive interactions between proteins and

PNIPAAM. This hydration shell is so strong, that it is still intact even at T = 40 °C, although

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it is known, that at this temperature PNIPAAM brush films lose considerable amounts of

water.[13]

The significant influence of the outermost PEL layer on the adsorbed HSA concentration is

illustrated in Fig. 9c, where the bound HSA amount at PEM terminated by polycations like

PEI (i.e., odd-numbered PEM layers) is compared to PEM terminated by polyanions such as

ALG or poly(acrylic acid) (PAC). Significantly, at PEM-8 of PEI/ALG and PEM-4 of

PEI/PAC, less HSA is adsorbed compared to PEM-9 of PEI/ALG and PEM-5 of PEI/PAC.

This can be explained by the anionic nature of HSA (isoelectric point IEP = 4.8) being bound

under either electrostatic attraction (odd PEM) or repulsion (even PEM). PAC seems to be

even more effective than ALG. Presumably, polysaccharidic ALG undergoes additional

interaction forces like H-bonding or van der Waals towards proteins.

3.2.2. Drug interactions on PEM films

Recently, PEM systems based on PEI and the polyanions ALG or PAC loaded with the

antiobiotic streptomycin (STRP) were reported.[25]

These model studies are relevant for the

functionalization of bone-substituting material by interfacial drug-eluting polyelectrolyte-

based material. PEM build-up, and STRP loading and release, was monitored using in-situ-

ATR-FTIR spectroscopy. Typical ATR-FTIR spectra of a PEM-PEI/ALG-8 loaded with

STRP (STRP/PEM: 1:5) in contact with water are shown in Fig. 10a. The (C-O) band of

STRP at approximately 1100 cm-1

can be used as a diagnostic marker for the STRP content

and the s(COO-) band at 1400 cm

-1 for the PEM film. The ratio between the former and the

latter was used to determine the percentage content of STRP (relative to the initial content).[25]

Fig. 10b shows release curves for PEI/ALG in comparison to PEI/PAC. Interestingly, a

smaller initial burst (IB, percentage of STRP released after 1 min) IB = 2% for PEI/ALG

compared to IB = 22% for PEI/PAC was obtained and also the residual amount (RA) of STRP

after 200 min was RA = 70 % for PEI/ALG and RA = 17% for PEI/PAC.

Since STRP has a tetrasaccharidic structure, we assume that it interacts stronger to the

polysaccharide-based ALG compared to synthetic PAC. Moreover, in Fig. 10b fits of the

ATR-FTIR release data to a simple Ritger/Peppas model[26]

according to A(t) = 100 - a tb were

performed, where A(t) is the integrated peak intensity of the (C-O) band.

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Fig. 10a. in-situ ATR-FTIR spectra from

PEM-8 films composed of PEI/ALG loaded

with STRP (STRP/PEM: 1:5) in contact with

water. The spectra are recorded as a function

of time (1- 240 min).

Fig. 10b. STRP release profiles from PEM-8

of PEI/ALG and PEI/PAC. (from [25]

with

kind permission of Elsevier).

The exponent b is a measure for the release dynamics in hydrogel-like films, in which b = 0.5

corresponds to diffusion release, b << 0.5 to dissolution and b >> 0.5 to hindered diffusion

drug release and a is an amplitude parameter. In our case, analysis reveals b = 0.54 for

PEI/ALG and b = 0.34 for PEI/PAC. Thus, for PEI/ALG STRP diffuses out of the PEM

phase. Hence, ATR-FTIR spectroscopy can be used as a simple initial screening method for

the detection of drug release from polymer films.

3. Conclusions

In this review, we presented an overview of the abilities of in-situ ATR-FTIR spectroscopy

for characterization of thin biorelated polymer films and their interactions with proteins and

drugs and its potential as a rapid screening method. Due to the small effective thickness (de)

of the ATR-FTIR method ranging up to a few multiples of 100 nm, depending on the

refractive indices of the IRE material (de decreases in the order Ge < Si< ZnSe), the strongly

absorbing water bands at 3700-3000 cm-1

((OH)) and around 1640 cm-1

((OH)) can be

conveniently compensated and phenomena at the solid/liquid interface are accessible. It was

shown that with the help of ATR-FTIR, one can obtain information about adsorption and

growth processes as well as chemical composition in polymer films as thin as a few

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nanometers. As FTIR spectroscopy in general is sensitive to internal or external hydrogen

bonding, especially to the carbonyl oxygen or the amide nitrogen, the intramolecular

interaction between related polymer segments or the intermolecular one to water in thin films

can be nicely studied by surface sensitive ATR-FTIR. These analytical options were

demonstrated in the examples of (i) reactive deposition of dopamine at inorganic model

surfaces, (ii) switching and adsorption of proteins on thermosensitive polymer brushes of

poly(N-isopropylacrylamide) (PNIPAAM) as well as (iii) adsorption of drugs to biorelated

polyelectrolyte multilayer (PEM). Sensitivity to polypeptide and protein conformation based

on diagnostic positions of the Amide I and Amide II band[27, 28]

is a further classical analytical

option for the FTIR method, which however is a topic of its own and was not treated herein.

Finally, limitations of the ATR-FTIR method should also be mentioned. These are mainly

related to the nonlinear relationship (damped exponential) between measured absorbance and

thickness d of thicker polymer films (d >> 300 nm). While for thin films (d < 300 nm), this

relationship can be approximated by a linear function (A(d) = k d) and processes at the

outermost zone of thin films are still observable by ATR-FTIR, for increasingly thick films,

the ATR-FTIR method becomes increasingly insensitive to processes occurring at the

outermost zone. Furthermore, by ATR-FTIR, from the measured absorbance A there is not a

direct access to the absolute value of the geometrical thickness d as it is possible by

ellipsometry, which however, is rather insensitive to the chemical composition. Thus, there is

a possibility based on the combination of ATR-FTIR and transmission-FTIR to access even

absolute values of the thickness of a polymer or organic film within a certain error range,

which will be the subject of a forthcoming paper.

Acknowlegements

Part of this work is related to the Transregio SFB TRR 79 (M7) of German Research

Foundation (DFG) "Materials for tissue regeneration in the systemically diseased bone"

(University Giessen, University Heidelberg, Technical University Dresden, and cooperating

research institutes) as well as DFG Grant IO 68/1-1.

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