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Colloids and Surfaces A: Physicochem. Eng. Aspects 343 (2009) 118–126

Contents lists available at ScienceDirect

Colloids and Surfaces A: Physicochemical andEngineering Aspects

journa l homepage: www.e lsev ier .com/ locate /co lsur fa

alcium uptake by casein embedded in polyelectrolyte multilayer

ilianna Szyk-Warszynskaa,∗, Csilla Gergelyb, Ewelina Jareka, Frédéric Cuisinierc,obert P. Sochaa, Piotr Warszynskia

Institute of Catalysis and Surface Chemistry PAS, ut. Niezapomianjek 8, 30-239 Krakow, PolandGroupe d’étude des semi-conducteurs Université Montpellier II, UMR 5650 CNRS, Place Eugéne Bataillon, 34095 Montpellier Cedex 05, FranceUFR Odontologie, Université Montpellier I, 545 Avenue prof. Viala, 34193 Montpellier Cedex, France

r t i c l e i n f o

rticle history:eceived 30 September 2008eceived in revised form 5 January 2009ccepted 30 January 2009

a b s t r a c t

The aim of our work was to investigate formation of polyelectrolyte multilayer films containing �- and�-casein. Since in neutral pH casein is negatively charged (as verified by electrophoretic mobility measure-ments) it has been used as a polyanionic layer for the film build-up. Casein containing films were formedon Si/SiO2 wafers. The formation of the film was investigated by liquid cell ellipsometry. After the multi-

vailable online 5 April 2009

eywords:aseinayer-by-layerolyelectrolyte films

layer films were formed they were contacted with solution containing calcium ions and changes in thefilm thickness were monitored. Additionally the surfaces of casein containing multilayers were analyzedwith AFM for the structural changes within the films occurring after binding of calcium ions. Presence ofcalcium ions bound in the film was also monitored by XPS. We concluded that casein embedded in thepolyelectrolyte multilayers preserves its ability to bind calcium ions.

alcium bindingultilayers

. Introduction

In recent years there has been an increased interest in intrinsi-ally unstructured proteins (IUP), i.e., proteins that in their naturaltate do not adopt stable, folded structures [1–4]. Proteins of thisype, abundant in nature, play an important role in living organisms.owever, despite their function is well understood, our knowledgeoncerning adsorption and self-organisation is rather limited. Aharacteristic feature of IUP’s is an open structure, which becomesreserved even after ligand binding. Casein is one of the most com-on IUP’s. It is a phosphoprotein present in mammalian milk and

ts products, where it occurs in a micellar form made of four majorypes [5,6]: �s1-, �s2-, �-, �-casein. �s1- and �s2-caseins comprise0% and 10%, respectively, of the whole casein content of milknd are usually called �s-caseins [7]. �-casein comprise 38% ofotal casein contents [8,9]. The function of caseins is to store andransport bio-available metal ions (especially, Ca(II) and Mg(II)) byequestering and transporting them from mother to the neonates2,7,10,11].

In aqueous solution casein is surface active [11–14] and forms

icellar aggregates. Single protein behaves as flexible, disordered,

olyelectrolyte-like molecule [15], therefore, it should be easilyntegrated into polyelectrolyte films. Casein has ability to bindalcium ions and therefore, it can be used in biotechnology and

∗ Corresponding author. Fax: +48 12 4251923.E-mail address: ncszyk@cyf-kr.edu.pl (L. Szyk-Warszynska).

927-7757/$ – see front matter © 2009 Elsevier B.V. All rights reserved.oi:10.1016/j.colsurfa.2009.01.038

© 2009 Elsevier B.V. All rights reserved.

in biomedical applications. Films containing caseins are used aspaints, which may be diluted with water or adhesives for labelingof glass containers [16]. Materials covered with casein containingfilms can be also applied in diary industry for the prevention ofcalcium deposit formation.

Molecular structures of two types of caseins used in our investi-gations are illustrated in Fig. 1. �(s1)-casein has molecular weight(MW) 23,000. The chain is build from 199 amino acids with 17proline residues. It has two hydrophobic regions, containing allthe proline residues, separated by a polar region, which con-tains all but one of eight phosphate groups and is highly charged.Molecular diameter of �(s1)-casein is 9 nm. �(s2)-casein: MW24,000; 207 residues, 10 prolines. Negative charges are concen-trated near N-terminus and positive charges near C-terminus. Bothcaseins can be precipitated at very low levels of calcium. Molec-ular weight of �-casein is 24,000. It has 209 residues and 35prolines. N-terminal region is highly charged, hydrophilic and C-terminal region is a hydrophobic. This amphiphilic protein actslike a detergent molecule. It is less sensitive to calcium precipi-tation. Its molecular diameter is 7.5 nm. MW of �-casein is 19,000,169 residues with 20 prolines. This casein is very resistant to cal-cium precipitation and it stabilizes other proteins and their micellaraggregates [20–22].

Proline acts as a structural disruptor in the middle of regular sec-ondary structure elements such as alpha helices and beta sheets.However, proline is also commonly found as a first residues of analpha helix and also in the edge strands of beta sheets. As prolinelacks a hydrogen on the amino group, it cannot act as a hydrogen

L. Szyk-Warszynska et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 343 (2009) 118–126 119

ski et

botsptpb

vttbawb[isa

ptectwnm

2

2

C(Hwrwp0

Fig. 1. Molecular models of � and �-casein derived by Thomas F. Kumosin

ond donor, only as a hydrogen bond acceptor. The large numberf proline residues in caseins causes particular bending of the pro-ein chain and inhibits the formation of closely packed secondarytructures [16]. Therefore, embedding of praline reach casein into aolyelectrolyte film should not effect to a large extend protein struc-ure. Most of the casein proteins in solution form micelles, which areolydisperse, roughly spherical aggregates with diameter rangingetween 150 and 300 nm [23].

Sequential adsorption of charged nanoobjects at interfaces is aery versatile technique to form nanostructured thin films. In par-icular the sequential adsorption of polyelectrolytes, also referredo as layer-by-layer adsorption technique which was introducedy Decher et al. [24,25], has attracted in the recent years muchttention. It has the advantage of producing multilayers filmsith well defined thickness and surface properties. The layer-

y-layer technique can be useful in wide range of applications26–28]. Embedding of proteins or other bio-active nanoparticlesn polyelectrolyte multilayer films can contribute to formation ofurface nanostructures [29], which can be used in a biomaterialrea.

The aim of our present work was to investigate formation ofolyelectrolyte multilayer films containing �- and �-casein ando verify if the casein embedded in the polyelectrolyte multilay-rs preserves its ability to bind calcium ions. The formation of theasein containing films was investigated by liquid cell ellipsome-ry. After the multilayer films were formed they were contactedith solution containing calcium ions and changes in the film thick-ess were monitored. Additionally the surfaces of casein containingultilayers were analyzed with AFM and XPS.

. Materials and methods

.1. Materials

�-casein (Cat. No. C6780-1G, min 70%) and �-casein (Cat. No.6905-1G, min 90%) from bovine milk, poly-l-lysine hydrochloridePLL), MW 30,000–70,000, (Cat. No. P-2636) and HEPES (N-2-

ydroxyethylpiperazine-N′-2-ethanesulfonic acid, Cat. No. H6147)ere obtained from Sigma. Sodium chloride pure p.a., hydrochlo-

ic acid, sodium hydroxide, sulfuric acid and hydrogen peroxideere obtained from POCh, Poland. Polished silicon wafers wereurchased from On Semiconductor Czech Republic, a.s. (Cz/100T-.5 mm/(100)/P Type).

al. [17,18] optimized using AMBER99 molecular mechanics force field [19].

2.2. Methods

Malvern NanoZeta dynamic light scattering (DLS) analyzer wasused for zeta potential and size measurement of �- and �-casein.They were measured in dependence of pH of casein solution. pHwas regulated by addition of HCl and NaOH. Zeta potential and sizewas measured also in presence of calcium ions. Concentration ofCa2+ was 2 and 50 mM.

Thickness of PLL/casein multilayer films were measured byimaging ellipsometer EP3 (Nanofilm). Ellipsometry is a sensitiveoptical technique that measures the change in the state of the polar-ization of light upon reflection from a planar interface in order togain information about its structure and optical properties. In anyellipsometry experiment, upon reflection of the polarised light ata known angle of incidence and with a known wavelength �, therelative change in the amplitude of the polarised light [expressedas tan( )] and the relative change in the phase difference betweenthe s- and p-components of the light (expressed as �) are deter-mined [30]. Fitting appropriate optical model to the measuredvalues of and �, thickness and optical properties (refractiveindex, absorbance) of the film adsorbed at surface are calculated[30].

We used ellipsometer equipped with liquid cell connected withsystem of syringes filled with appropriate solution needed forexperiments (Fig. 2). In our case the wavelength of laser light was�= 532 nm. The liquid cell was illuminated under the angle of inci-dence (AOI) 60◦. For dry samples AOI = 75◦ was used.

To build-up multilayer films with casein and PLL we used layer-by-layer technique [24,25], schematically presented in Fig. 3.

The multilayer films were deposited on a silicon wafers Si/SiO2.Before using as support for the films, wafers were washed in piranhasolution (H2SO4:H2O2, 1:1), boiled three times in distilled waterand rinsed with excess of distilled water. To form polycation layer100 ppm PLL solution in HEPES (10 mM, 0.15 M NaCl, pH 7) was used.PLL as a synthetic polypeptide, consists of the amino acid-lysine,which has an amine group at the end of the side-chain and therefore,it is positively-charged at neutral pH. As a polyanion �- or �-casein(0.1 g/l in HEPES) solution was used. Washing steps were realizedby using HEPES buffer.

Multilayer films containing five layers—(PLL/CAS)2PLL and sixlayers (PLL/CAS)3 were formed on surface of silicon wafers. After-wards, wafers with multilayer films terminated with casein or withPLL were immersed in CaCl2 solutions. Concentration of CaCl2 was2, 20 and 50 mM. Such prepared plates were dried and analyzed

120 L. Szyk-Warszynska et al. / Colloids and Surfaces A: P

Fig. 2. Schematics of the liquid cell ellipsometry experiment.

F

b(TwmIo

fitu

ig. 3. Illustration of the principle of formation of multilayer films containing casein.

y ellipsometry in order to measure the thickness of adsorbedPLL/CAS) films and its change upon contacting with calcium ions.he multilayer (PLL/CAS) films were also formed directly on Si/SiO2afers in the liquid cell, so adsorption of PLL and casein could beonitored “in situ” and thickness of multilayer film was measured.

n such a way the kinetic adsorption of multilayer film could be

btained.

X-ray photoelectron spectroscopy (XPS) was applied to con-rm presence of calcium in the (PLL/CAS) multilayer. To performhe analysis, the samples were evacuated to the ultrahigh vac-um (UHV, 3 × 10−10 mbar). Then un-monochromatized Al K�

Fig. 4. Dependence of zeta potential of �-casein (a) and �-casein

hysicochem. Eng. Aspects 343 (2009) 118–126

X-ray source (1486.6 eV) was applied to generate core excitation.Binding energy of the excited photoelectrons was measured withhemispherical analyzer (SES R 4000, Gammadata Scienta). Thespectrometer was calibrated according to ISO 15472:2001. Thedepth of XPS analysis was estimated to 12 nm.

Topology of surfaces of PLL/casein films were analyzed in drystate with NT-MDT Atomic Force Microscope working in the contactmode.

3. Results and discussion

The pH dependencies of zeta potential of �- and �-casein mea-sured in aqueous solution are shown in Fig. 4. One can see thatboth caseins � and � are positively charged in acid solution andthey are negatively charged at neutral and basic conditions. Theisoelectric point of �- and �-casein is observed around pH 5–5.5.This is in a good correlation with the data found in literature [31,32].For studying adsorption of �- and �-casein at the polyelectrolytefilms the pH 7 was chosen. Caseins are then negatively charged andcan be used as polianions in the conditions close to physiologicalones.

In Table 1 data concerning zeta potential and size of caseinmicelles are collected. The measurements were done without andin presence of calcium ions. One can see that addition of calciumions increases the value of zeta potential of caseins (makes it lessnegative). Simultaneously, it causes the increase of the size of caseinmicelles. This size is in agreement with one found in literature [33]and ranges from 40 nm to over 500 nm. In presence of very highconcentration of calcium ions (50 mM CaCl2) both �- and �-caseincreate aggregates, which size is between 1.2 �m even to 3.2 �m.This phenomenon is observed in both aqueous solution and inHEPES, however, the sizes of micelles in HEPES are smaller thanin water.

In Fig. 5 the thickness of multilayer films (PLL/CAS) in depen-dence of number of adsorbed layer is shown. Data were obtainedfrom ellipsometry measurements of film thickness on dried Si/SiO2plates. The refractive index of n = 1.55 of the film was independentlydetermined by ellipsometry multi-angle analysis. It can be seenthat multilayers build with �-casein form thicker films that thosecreated with �-casein. The increase of film thickness, which cor-respond to adsorption of casein layer, is higher than one for PLLand is in agreement with casein molecular size. For example – anincrease of film thickness equal to 8 nm (Fig. 5a, for second layer of�-casein) corresponds to the molecular size of �-casein. The sametrend can be observed for adsorption of �-casein on PLL. Therefore,

the ellipsometry results presented in Fig. 5 give evidence that mul-tilayer films with PLL and casein can be formed. After immersion ofthe films in the solutions containing calcium ions, further growthof the film thickness can be observed. One can argue that the filmsended with casein (Fig. 5a and c) adsorbed more of calcium ions

(b) solution on pH. Concentration of casein was 0.03 g/dm3.

L. Szyk-Warszynska et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 343 (2009) 118–126 121

Table 1Zeta potential and size of casein micelles in solutions of CaCl2.

Casein in water Casein in HEPES 10 mM pH 7, 0.15 M NaCl

ccas (g/l) cCaCl2 (mM) Zeta pot. (mV) Size (nm) ccas (g/l) cCaCl2 (mM) Zeta pot. (mV) Size (nm)

�-casein0.01 0 −32.7 87 0.01 0 −13.5 41

2 −20.0 231 2 −8.0 5050 −4.6 1473 50 −4.6 474

0.03 0 −40.0 154 0.03 0 −10.9 712 −15.1 215 2 −9.3 188

50 −5.0 1435 50 −6.2 400

0.1 0 −48.2 142 0.1 0 −11.4 2342 −17.7 147 2 −9.4 74

50 −5.0 3183 50 −5.3 2025

�-casein0.01 0 −22.9 153 0.01 0 −11.4 415

2 −13.8 169 2 −6.3 55350 −4.5 319 50 0.3 660

0.03 0 −14.8 161 0.03 0 −10.6 2512 −14.1 151 2 −9.5 530

50 −3.3 216 50 −5.5 1281

0

tfi

a“mafs

F(

.1 0 −16.2 1892 −14.7 192

50 −3.9 196

hat those ended with PLL (Fig. 5b and d), since larger increase oflm thickness is observed.

Fig. 6 illustrates variations of the ellipsometric parameters �nd during formation of (PLL/�-casein) multilayer film on Si/SiO2

in situ” in the liquid cell. Line perpendicular to time axis indicateoments of start of injection of respectively PLL, HEPES, �-casein

nd CaCl2 solution into the liquid cell. Every injection of the solutionor the rinsing step or adsorption of consecutive layer was made noooner than the ellipsometric parameters, � and , reached con-

ig. 5. Dependence of thickness of dry (PLL/casein)n films on Si/SiO2 wafer build-up by Lbc) and (d) multilayer films containing �-casein). Concentration of �- and �-casein was 0

0.1 0 −16.4 492 −8.3 372

50 −5.1 302

stant values after the previous adsorption step. From these plateauvalues of ellipsometric parameters the thickness of the adsorbedlayer was calculated using three layer optical model (Si/SiO2/film).The refractive index of n = 1.5 was used for the (PLL/CAS) film in

HEPES solution.

One can see in Fig. 6 that the ellipsometric parameters changeafter each injection of either PLL or casein, however, the change ofellipsometric signal was more pronounced during casein adsorp-tion step. At the last stage of experiment, the (PLL/CAS)2PLL film

L method on the number of layers. (a) and (b) multilayer films containing �-casein,.1 g/dm3 in HEPES, cHEPES = 10 mM, 0.15 M NaCl, pH 7.

122 L. Szyk-Warszynska et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 343 (2009) 118–126

Fig. 6. Adsorption kinetics of (PLL/�-CAS) film as monitored by the liquid cell ellip-s0i

wic

(kdt

third layer of PLL (see Fig. 7a). Similar observation concerned films

Fe

Fe

ometry. Solid line correspond to� and dashed line to parameter. cHEPES = 10 mM,.15 M NaCl, pH 7, cPLL = 0.1 g/l in HEPES, c�-cas = 0.1 g/dm3 in HEPES, cCaCl2 = 20 mM

n H2O.

as exposed to the solution of CaCl2. One can observe that afternjection of solution calcium ions the signal of � and did nothange significantly.

The results of the mathematical treatment with the constant

n,k) optical model of the ellipsometric signal obtained duringinetic experiments data are shown in Fig. 7. Similarly as forried (PLL/CAS) films obtained by classic layer-by-layer dippingechnique, the big increase (ca. 10 nm) of the film thickness after

ig. 7. Dependence of thickness of (a) (PLL/�-CAS)n-CaCl2 and (b) (PLL/�-CAS)n-CaCl2 filmxperimental conditions as in Fig. 6.

ig. 8. Dependence of thickness of (a) (PLL/�-casein)3-CaCl2 and (b) (PLL/�-casein)3-Cllipsometric cell. Concentration of �- and �-casein was 0.1 g/dm3 in HEPES 10 mM, pH 7

Fig. 9. X-ray Ca 2p core excitation of the (PLL/�-casein)3 film without and with CaCl2treatment.

adsorption of casein on PLL layer is also observed “in situ”. Thatincrease, corresponding to the molecular size of casein, proves againability of formation of casein layer on PLL terminated film. Contrarywhat was observed for dried films, the thickness of wet (PLL/�-CAS) film did not increase after adsorption of calcium ions on the

with �-casein (Fig. 7b). When the multilayer film, with casein layeron top of it, is contacted with the solution of calcium ions only asmall increase of the thickness of (PLL/casein) film is observed (seeFig. 8a and b). So one can conclude that ellipsometric measure-

s on number of layers. Measurement were perform in liquid ellipsometric cell. The

aCl2 films on number of deposited layers. Measurement were perform in liquid, 0.15 M NaCl, concentration of PLL 100 ppm in HEPES, cCaCl2 = 20 mM.

L. Szyk-Warszynska et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 343 (2009) 118–126 123

Table 2Ellipsometric thickness of wet and dry (PLL/CAS) films without and with contact with CaCl2 solution.

Layer �-casein �-casein

Dry films thickness (nm) Wet films thickness (nm) Dry films thickness (nm) Wet films thickness (nm)

PLL 1.0 1.0 0.3 1.6PLL/CAS 1.7 14.4 1.9 9.1PLL/CAS/PLL 2.2 17.9 2.2 13.0(PLL/CAS)2 5.4 27.5 5.0 23.1(PLL/CAS)2PLL 9.7 30.8 6.5 25.4CaCl2 2 mM on (PLL/CAS)2PLL 10.0 6.1CaCl2 20 mM on (PLL/CAS)2PLL 29.5 24.8CaCl2 50 mM on (PLL/CAS)2PLL 11.8 7.4(PLL/CAS)3 16.4 33.8 9.4 30.4CaCl2 2 mM on (PLL/CAS)3 20.7 10.4CaCl2 20 mM on (PLL/CAS)3 34.1 31.3CaCl2 50 mM on (PLL/CAS)3 22.4 10.9

Table 3Atomic concentration of Ca, Si and Ca/C atomic ratios obtained from XPS measurements for different Si/SiO2/(PLL/CAS) systems.

Sample Ca (at.%) Ca/C Si (at.%) Ca after 4 h rinsing (at.%) Ca/C after 4 h rinsing

(PLL/�CAS)3 3.8 0.060 2.4 0.09 0.0039(PLL/�CAS)2/PLL 7.1 0.138 10.3 0.02 0.0004(PLL/�CAS)3 2.5 0.043 7.8 0.03 0.0006(PLL/�CAS)2/PLL 3.0 0.067 21.3 0.00 0

Fig. 10. AFM picture of (PLL/�-casein)3 film treated with CaCl2 adsorbed on Si/SiO2 plate; (a) AFM picture; (b) topological image; (c) z-profile. Film formed in the ellipsometricliquid cell. C�-cas = 0.1 g/dm3 in HEPES 10 mM, pH 7, 0.15 M NaCl, cCaCl2 = 20 mM.

124 L. Szyk-Warszynska et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 343 (2009) 118–126

F film tr( 10.

mc�fi

tw(TfiscctCvotftc

i

ig. 11. Close-up of the example of large casein aggregate found in (PLL/�-casein)3

c) z-profile. Film formed in the ellipsometric liquid cell in the conditions as in Fig.

ents of film thickness in solution does not give firm evidence ofalcium uptake in the casein containing film and maintaining of- and �-casein bioability after being embedded in the multilayerlm.

Comparing data from Fig. 5 for thickness of dry films withhe corresponding data shown in Figs. 7 and 8 for thickness ofet (PLL/casein) films one can see that the wet films formed by

PLL/casein) multilayers are much thicker than dried ones (seeable 2). This observation concerns both �- and �-casein containinglms. We can suspect that because of swelling due to hydration, thetructure of the wet films is less compact than dry films [34] andalcium ions can easily penetrate the space between the PLL andasein chains. So however, we did not see the significant increase ofhe thickness of wet films after adsorption of calcium ions, in facta2+ ions were bind to the casein. Lose structure of the film pro-ides enough space to accommodate any conformational changesf casein due to calcium binding, without influencing the filmhickness. Upon drying of calcium containing films, their recon-

ormation accompanied by CaCl2 crystallisation can occur, leadingo the increase of film thickness in comparison with ones withoutalcium.

To prove our hypothesis concerning the presence of calcium ionsn the (PLL/casein) multilayers we made XPS analysis of the films

eated with CaCl2 adsorbed on Si/SiO2 plate; (a) AFM picture; (b) topological image;

formed during the liquid cell ellipsometry experiments. Two seriesof the samples were analysed. One series were the Si/SiO2 plateswith the films adsorbed, directly taken from the liquid ellipsometriccell and shortly rinsed in distilled water to remove buffer solu-tion and excess of salt. Then the samples were evacuated to UHVconditions, exposed to X-ray and the photoelectron spectra wereacquired. The second series of the (PLL/CAS) films, after their for-mation in the liquid cell was conditioned in excess of distilled waterfor 4 h and then studied with XPS. All XPS measurements confirmedpresence of calcium ions in (PLL/CAS)-type layers when they werecontacted with CaCl2 solution (see Fig. 9 as example of XPS spectra).Selected results of the elemental analysis for 12 nm thick interfaciallayer are given in Table 3. They demonstrated that, in the films, mostof calcium in the films is present as CaCl2 that cannot be removedduring the typical rinsing process. The film micellisation of caseinand crystallisation of CaCl2 occurs upon drying the film, that leadsto the increase of dry film thickness. After a prolonged soaking ofthe films in water, concentration of Ca in the film decreases by 2

orders of magnitude but the remaining calcium is bound to thecasein since no chlorine is detected in the XPS spectrum. In agree-ment with observed ability of calcium binding of two types of caseinin solution, calcium concentration in films containing �-casein washigher than in films with �-casein. The (PLL/�-CAS)2PLL multilayers

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L. Szyk-Warszynska et al. / Colloids and Surface

ashed for 4 h in water, showed lower Ca content than the (PLL/�-AS)3 ones, due to lower amount of casein adsorbed in the film. Inhe case of (PLL/�-CAS)2PLL film, the amount of adsorbed Ca2+ waselow the level of detection. Contrary, in the sample taken directlyrom liquid cell, the (PLL/�-CAS) layer terminated with PLL showedigher Ca content than obtained for (PLL/�-CAS)3 ones (see col-mn 1 in Table 3). The possible reason for this observation was thataCl2 can be easily removed from the top casein layer during thehort rinsing process. The analysis of Ca/C ratios (Table 3), in case ofntreated and washed up samples, corroborates above conclusions.

Taking into account the XPS analysis depth of 12 nm, the dataor silicon concentration in the layer collected in Table 3 confirmllipsometric findings that films built with �-casein are thickerhan ones with �-casein (lower Si concentration). The same obser-ation concerns the thickness difference between (PLL/CAS)2PLLnd (PLL/CAS)3 multilayer structures. Additional experiments per-ormed on the multilayer formed by layer-by-layer techniqueutside the ellipsometric cell confirmed these results.

To examine the details of surface of casein containing multilay-rs, the silicon wafers with (PLL/�- or �-casein) films were alsonalysed by AFM microscopy. In Fig. 10 the example of the AFMmage of (PLL/�-casein)3 film treated with CaCl2 is shown. Thei/SiO2 plate with PLL/casein film was first analysed by ellipsome-ry in the liquid cell and then after drying used to obtained its AFMmage. We can observe numerous small casein micelles on the sur-ace of the silicon wafer, accompanied by big aggregates. The size ofhe big aggregates is around few micrometers with the “shell” thick-ess of hundred nanometres (conf. Fig. 11). The micelles has theize ranging from few to hundreds of nanometers with the averageoughness of micelles covered surface of 25 nm. Those sizes corre-pond to ones observed in casein solutions by DLS (see Table 1). Thatan be considered as additional evidence that calcium ions bind tohe casein embedded in multilayer (PLL/CAS) film.

. Conclusions

Sequential adsorption of polyelectrolytes and proteins can be auitable technique for formation of surface nanostructures, whichay be used in a biomaterial area. Therefore, in present worke concentrated on formation of polyelectrolyte multilayer films

ontaining �- and �-casein. We found that both caseins were neg-tively charged in neutral (and basic) conditions, therefore, theyan be used, together with biotolerable polycation poly-l-lysineydrochloride (PLL), to construct polyelectrolyte/protein multi-

ayer. Measuring the ellipsometric thickness of the multilayers “initu” during their formation on silicon wafers in the liquid cell, weound that the larger increase of film thickness can be attributedo adsorption of casein. This increase was in quantitative agree-

ent with the molecular size of casein used and the multilayertructures containing �-casein exhibited larger thickness than onesith �-casein. The same trends were observed when multilayerlm thickness of dry structures formed by layer-by-layer depositionethod was measured.After formation of multilayer films, they were contacted with

he solution of CaCl2 and the changes of thickness were mon-tored. Whereas, the increase of film thickness, which could bettributed to binding of calcium by casein, was detected when drylms were measured, thickness of films formed “in situ” in the liq-id cell and contacted with CaCl2 remained practically unchanged.o resolve that controversy in the results, we analyzed with XPS the

PLL/casein) multilayers, formed during the liquid cell ellipsometryxperiments, for presence of bound calcium. These measurementsonfirmed presence of calcium ions in the casein containing mul-ilayers when they were contacted with CaCl2 solution. Most ofalcium was present in the film as CaCl2, which could not be rinsed

[

[

hysicochem. Eng. Aspects 343 (2009) 118–126 125

away by applying a simple rinsing procedure. Prolonged soaking offilms in the distilled water led to removal of calcium chloride butcalcium bound to casein could still be detected. We found that cal-cium concentration in films containing �-casein was higher thanin films with �-casein. That remains in agreement with generallyaccepted observation concerning ability of calcium binding of thesetwo types of casein in solution. We found that (PLL/CAS)2PLL multi-layers showed lower content of bound Ca than the (PLL/CAS)3 ones,due to lower amount of casein adsorbed in the film. Combiningthe results obtained by ellipsometry and XPS we can conclude, thatcasein embedded in the polyelectrolyte multilayers preserves itsability to bind calcium ions. Lack of increase of wet (PLL/casein)film thickness when exposed to calcium ions can be explained byloose structure of hydrated multilayer providing enough space toaccommodate any conformational changes of casein due to calciumbinding, without influencing the film thickness. Upon drying ofcalcium containing films their reconformation accompanied withcalcium crystallisation can occur, leading to the increase of filmthickness in comparison with ones without calcium.

Analysis of topography of calcium containing (PLL/casein) mul-tilayers deposited on silicon wafer by AFM, indicated presence ofcasein micelles and large aggregates, which sizes corresponded toones observed in casein solutions. That can be considered as addi-tional evidence of calcium ions binding to the casein embedded inmultilayer films.

Acknowledgments

ECO-NET 2007 international cooperation project betweenFrance, Hungary and Poland and MNiSW grant 81/N-ECO-NET/2007/0 for financial support. Joanna Piekoszewska and AnnaTrybała for help with ellipsometry measurements and Dr. JakubBarbasz for performing AFM measurements.

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