pubs.acs.org/Langmuir

Assembly of Purple Membranes on Polyelectrolyte Films

Marie-belle Saab,† Elias Estephan,† Thierry Cloitre,† Ren�e Legros,† Fr�ed�eric J. G. Cuisinier,‡

L�aszl�o Zim�anyi,‡,§ and Csilla Gergely*,†

†Groupe d’Etude des Semi-conducteurs, UMR 5650, CNRS-Universit�e Montpellier II, 34095, MontpellierCedex 5, France, ‡EA 4203, UFR Odontologie, Universit�e Montpellier I, 34193 Montpellier Cedex 5, France,

and §Institute of Biophysics, Biological Research Center of the Hungarian Academy of Sciences, H-6701Szeged, Hungary

Received September 22, 2008. Revised Manuscript Received February 9, 2009

The membrane protein bacteriorhodopsin in its native membrane bound form (purple membrane) was adsorbed andincorporated into polyelectrolyte multilayered films, and adsorption was in situ monitored by optical waveguide light-mode spectroscopy. The formation of a single layer or a double layer of purple membranes was observed when adsorbedon negatively or positively charged surfaces, respectively. The purplemembrane patches adsorbed on the polyelectrolytemultilayers were also evidenced by atomic force microscopy images. The driving forces of the adsorption process wereevaluated by varying the ionic strength of the solution as well as the purple membrane concentration. At high purplemembrane concentration, interpenetrating polyelectrolyte loops might provide new binding sites for the adsorption of asecond layer of purple membranes, whereas at lower concentrations only a single layer is formed. Negative surfaces donot promote a second protein layer adsorption. Driving forces other than just electrostatic ones, such as hydrophobicforces, should play a role in the polyelectrolyte/purple membrane layering. The subtle interplay of all these factorsdetermines the formation of the polyelectrolyte/purple membrane matrix with a presumably high degree of orientationfor the incorporated purple membranes, with their cytoplasmic, or extracellular side toward the bulk on negatively orpositively charged polyelectrolyte, respectively. The structural stability of bacteriorhodopsin during adsorption onto thesurface and incorporation into the polyelectrolyte multilayers was investigated by Fourier transform infrared spectro-scopy in attenuated total reflection mode. Adsorption and incorporation of purple membranes within polyelectrolytemultilayers does not disturb the conformational majority of membrane-embedded R-helix structures of the protein, butmay slightly alter the structure of the extramembraneous segments or their interaction with the environment. This highstability is different from the lower stability of the predominantly β-sheet structures of numerous globular proteins whenadsorbed onto surfaces.

Introduction

The interactions between biological macromolecules and solidsurfaces are of primary importance in numerous fields ofmaterialsciences and in the design of new coatings for biomaterials.Protein adsorption on surfaces is a complex process, which isgoverned by the hydrophobicity and hydrophilicity of the sur-face,1 the surface charge,2 roughness,3 and free energy.4,5 Directadsorption of a protein onto a surface often induces loss of thefunctional activity and denaturing of the adsorbed protein. Theuse of underlying polyelectrolyte films (PEFs) may offer con-venient solutions for the problems encountered due to directanchoring of proteins to bare surfaces. PEFs are built up by thealternated adsorption of polycations and polyanions from aqu-eous solution at a solid/liquid interface.6,7 Due to this layer-by-layer construction, all PEFs exhibit excess charges, alternativelypositive and negative, on their surfaces. These excess charges are

the motor of their buildup, and also facilitate the adsorption of agreat variety of compounds on the PEF surface.8-10

Previously, it was shown that proteins may adsorb on eitherpositively or negatively charged polyelectrolyte (PE)-terminatingfilms.11,12 However, the ionic strength, pH, and the chargedifference between the adsorbed protein and terminating filmlayer influence the thickness and the amount of protein taken up.These physicochemical parameters provide a fine-tuning in theproperties of the obtained multilayered films. A PEF coating canalso preserve the secondary structure of adsorbed proteins.13,14

Therefore, optimization and characterization of the applied PEFsare essential for each application. It is certainly due to theirnumerous tunable properties that the use of various multilayeredPEFs has been greatly extended in biomaterials science.

The aim of our work is to use PEFs as a scaffold to produce anordered matrix incorporating membrane proteins in an orientedway within the PE layers. To this end, the adsorption ofbacteriorhodopsin (BR) in its membrane-bound form (purple

*Corresponding author. Address: Groupe d’Etude des Semiconducteurs,UMR 5650, CNRS-Universit�e Montpellier II, Pl. Eug�ene Bataillon 34095Montpellier Cedex 5, France. Tel: 0033467143248. Fax: 0033467143760.E-mail: Gergely@ges.univ-montp2.fr.(1) Grinnell, F.; Feld, M. K. J. Biol. Chem. 1982, 257, 4888–4893.(2) Qiu, Q.; Sayer, M.; Kawaja, M.; Shen, X.; Davies, J. E. J. Biomed. Mater.

Res. 1998, 42, 117–127.(3) Dufrene, Y. F.; Marchal, T. G.; Rouxhet, P. G. Langmuir 1999, 15, 2871–

2878.(4) Absolom,D. R.; Zing,W.; Neumann, A.W. J. Biomed.Mater. Res. 1987, 21,

161–171.(5) Norde, W.; Lyklema, J. Biomater. Sci. Polym. Ed. 2 1991, 183-202.(6) Decher, G.; Hong, J. D.; Schmitt, J. Thin Solid Films 1992, 210, 831–835.(7) Decher, G. Science 1997, 277, 1232–1237.

(8) Picart, C.; Lavalle, P.; Hubert, P.; Cuisinier, F. J. G.; Decher, G.; Schaaf, P.;Voegel, J.-C. Langmuir 2001, 17, 7414–7424.

(9) Gergely, C.; Bahi, S.; Szalontai, B.; Flores, H.; Schaaf, P.; Voegel, J.-C.;Cuisinier, F. J. G. Langmuir 2004, 20, 5575–5582.

(10) Gergely, C.; Szalontai, B.; Moradian-Oldak, J.; Cuisinier, F. J. G. Bioma-cromolecules 2007, 8, 2228–2236.

(11) Ladam, G.; Gergely, C.; Senger, B.; Decher, G.; Voegel, J.-C.; Schaaf, P.;Cuisinier, F. J. G. Biomacromolecules 2000, 1, 674–687.

(12) Picart, C.; Ladam, G.; Senger, B.; Voegel, J.-C.; Schaaf, P.; Cuisinier, F. J.G.; Gergely, C. J. Chem. Phys. 2001, 115, 1086–1094.

(13) Caruso, F.; Mohwald, H. J. Am. Chem. Soc. 1999, 121, 6039–6046.(14) Schwint�e, P.; Voegel, J.-C.; Picart, C.; Haikel, Y.; Schaaf, P.; Szalontai, B.

J. Phys. Chem. B. 2001, 105, 11906–11916.

Published on Web 3/24/2009

© 2009 American Chemical Society

DOI: 10.1021/la9002274Langmuir 2009, 25(9), 5159–5167 5159

membrane, PM) on charged surfaces of PEs was studied underdifferent physicochemical conditions in order to explore thevarious interactions at the base of the adsorption of membraneproteins.

PM patches are localized in the cell membrane of Halobacter-ium salinarum, and consist of the sole protein BR and lipids in a75-25% (w/w) composition.15 BR is a seven-transmembrane R-helical protein with a covalently bound retinal chromophore. Itsfunction is light-driven transmembrane proton pumping againstthe proton electrochemical gradient across the cell membrane,and thereby conserving free energy for ATP synthesis. The X-raystructure of BR is known at atomic resolution,16 and most of themolecular details of its proton transporting photocycle have beenelucidated. BR is also one of the most promising candidates bothin solubilized form and in PM for biomaterial sciences applica-tions because of its enormous stability and favorable opticalproperties. Because of its unique optical properties, numerousapplications using BR as an integrated optical switch are alsoforecasted.

PM possesses a permanent electric dipole moment17 and there-fore can be oriented in an electric field. The static and the dynamicresponse of optical waveguides coated with a dried PM film havebeen reported previously.18-20 In these works, PMwas depositedby evaporation ofwater from the protein solution, thus no controlover the thickness or orientation was assured, and inhomogene-ities in the thickness of the obtained BR film were observed.20

Transient absorption and photovoltage studies of self-assembledPM/polycation multilayer films have been previously reported,21

but without the aim of structural and optical characterization ofthe obtained architectures. In this sense, new solutions forlayering BR in its membrane bound form in a controlled way,onto any substrate, as well as incorporating them in a matrix witha high local organization, could be of wide interest.

Here we report on the adsorption of PMs on and into PEmultilayered films produced by the deposition of polyethyl-eneimine (PEI) followed by the alternating physisorption ofanionic polystyrene sulfonate (PSS) and cationic polyallylamine(PAH). The step-by-step buildup of the multilayers and the PMadsorption were monitored in terms of the thickness and therefractive index by means of optical waveguide light-modespectroscopy (OWLS). No changes in the secondary structureof the membrane-bound BR as a consequence of its insertion intoPEF architectures were observed by Fourier transform infraredspectroscopy in attenuated total reflection mode (FTIR-ATR).Atomic force microscopy (AFM) was employed to visualize thesurface of the PEFs and the layered PMs. These detailed pieces ofstructural information on the PE/BR matrices could be interest-ing in further photoelectric and photochromic applications ofthis material.

Materials and Methods

Materials. Anionic poly(sodium 4-styrenesulfonate) (PSS,MW= 60 000), cationic poly(allylamine hydrochloride) (PAH,

MW= 70000), and cationic poly(ethyleneimine) (PEI, MW=60 000) were purchased from Aldrich. NaCl (purity 99.5%) waspurchased from Fluka; tris-(hydroxymethyl)-aminomethane(TRIS) and 2-(N-morpholino)ethanesulfonic acid (MES) werefrom Sigma. All the chemicals of commercial origin were usedwithout further purification. Ultrapure water (Milli-Q plussystem, Millipore) was used for solutions and in the differentcleaning steps. All buffer solutions were degassed under vacuumand filtered before use. The PEs were dissolved in 25 mMMES,25 mM TRIS, and 100 mM NaCl (pH 7.4) buffer (0.15 M ionicstrength) at a concentration of 5mg/mL. Some experiments wereperformed at three different ionic strengths (0.05, 0.15 and 0.55M) by varying the concentration of NaCl in the buffer (0, 0.1,and 0.5M, respectively). PMs were isolated fromHalobacteriumsalinarum following standard procedures.13 PM were suspendedin the same buffer as the PEs. The PM concentration is referredto by the corresponding protein concentrations of 30 μM and150 μM, determined using the visible and near UV extinctions ofBR. PEFs were built up by sequential physisorption of PSS andPAHonto a precursor layer of PEI, and all adsorption stepswereseparated by rinsing.Experimental Methods. Optical Waveguide Light-

Mode Spectroscopy. The buildup of multilayered PEFs andprotein adsorption onto PEFs were followed in situ by OWLS,an optical technique allowing in situ study of the adsorption ofmacromolecules onto a substrate.22 The technique is based onthe confinement of a laser beam in a planarwaveguide formed bya high refractive index layer (n ∼ 1.7) coated onto a glasssubstrate.22,23 For a discrete coupling angle, the laser beam iscoupled into the waveguide via a diffraction grating and thepropagation of the totally reflected light is monitored in realtime.Whenmaterial adsorbs, it perturbs the evanescent field andleads to changes in the effective refractive index of the guidedtransverse electric (NTE) and magnetic (NTM) modes, highlysensitive to the changes of the refractive indices. OWLS recordsvariations in NTE and NTM with high precision (ΔN ∼ 10-5)roughly up to a film thickness of 350 nm, corresponding to halfof the wavelength of the laser.24 By measuring the two modessimultaneously, then solving the mode equations,12,24 the struc-tural parameters of the adsorbed layers, i.e., refractive index andthickness (nA,dA) were obtained. OWLS data were analyzed byassuming that the multilayers behave as homogeneous andisotropic films. Anisotropy cannot be excluded for dry and/orthin films as recently reported.25 All our experiments wereperformed in a liquid cell, and the rather thick PEFs were builtup from aqueous solutions. Earlier scanning angle reflectometrymeasurements on PSS-PAH wet films were successfully ana-lyzed by the optical invariants method and evidenced that theselayers can be considered homogeneous and isotropic.12,24

The buildup of the PE multilayer film was performed asfollows. First, the PEI solution (5 mg/mL) was injected intothe cell and left to adsorb for 20 min. Then, in the same way,architectures of PEI-PSS, PEI-PSS-PAH, and PEI-(PSS-PAH)2 were built progressively. PE adsorption was alwaysseparated by a 20min long rinsing step to remove excessmaterialfrom the measuring cell. PMs were then adsorbed onto thesemultilayer films and allowed to adsorb from a continuous flowthrough the measuring cell (4 mL/h) for about 1 h. Once theoptical signals leveled off, the protein solution was replaced bybuffer solution and desorption of the adsorbed protein (if any)was monitored. The experiments (both PEF construction andPM adsorption) were performed at various ionic strengthsusing three different concentrations of NaCl: 0.05 M buffer(0MNaCl, 0.025MMES, 0.025MTRIS), 0.15M buffer (0.1MNaCl, 0.025 MMES, 0.025 M TRIS), and 0.55 M buffer (0.5 M

(15) Oesterhelt, D.; Stoeckenius, W. Methods Enzymol. 1974, XXXI, 667–678.(16) H. Luecke, B.; Schobert, H. T.; Richter, J.; Cartailler, P.; Lanyi, J. K. J.

Mol. Biol. 1999, 291, 899–911.(17) Barabas, K.; Der, A.; Dancshazy, Zs.; Ormos, P.; Keszthelyi, L; Marden,

M. Biophys. J. 1983, 43, 5–11.(18) Ormos, P.; Fabian, L.; Oroszi, L.; Wolff, E. K.; Ramsden, J. J.; Der, A.

Appl. Phys. Lett. 2002, 80, 4060–4062.(19) F�abi�an, L.; Oroszi, L.; Ormos, P.; D�er, A. In Molecular Electronics: Bio-

sensors and Bio-computers; Barsanti, L. Ed.; Kluwer Academic Publishers:Dordrecht/Boston/London, 2003; p 341.(20) Lukacs, A.; Garab, G.; Papp, E. Biosens. Bioelectron. 2006, 21, 1606–1612.(21) Jussila, T.; Li, M.; Tkachenko, N. V.; Parkkinen, S.; Li, B.; Jiang, L.;

Lemmetynien, H. Biosens. Bioelectron. 2002, 17, 509–515.

(22) Tienfenthaler, K.; Lukosz, W. J. Opt. Soc. Am. B 1989, 6, 209–215.(23) Ramsden, J. J. J. Mol. Recogn. 1997, 10, 109–120.(24) Picart, C.; Gergely, C.; Arntz, Y.; Voegel, J.-C.; Schaaf, P.; Cuisinier, F. J.

G.; Senger, B. Biosens. Bioelectron. 2004, 20, 553–561.(25) Horvath, R.; Ramsden, J. J. Langmuir 2007, 23, 9330–9334.

DOI: 10.1021/la9002274 Langmuir 2009, 25(9),5159–51675160

Article Saab et al.

NaCl, 0.025 M MES, 0.025 M TRIS). PM was adsorbed ontoeither positive (PAH) or negative (PSS) PE-terminated films.After the last PM adsorption step, the buildup of PE layers withalternating charge was continued by first applying the same PEas the last one before PM.

Atomic Force Microscopy. AFM images of the PE/proteinlayers were recorded in buffer, with an Asylum MFP-3D headand Molecular Force Probe 3D controller (Asylum Research,Santa Barbara, CA). Height and phase images were taken intapping mode using silicon nitride and rectangular cantilevers(Olympus Microcantilever, OMCL-BL-RC150VB) at a drivefrequency of 18 KHz; only the height images are reported. Wealways performed several scans over a given surface areaassuring reproducible images. Typically 512 � 512 pointscans were taken at 1 Hz scan rate. Images were first recordedon 20 � 20 μm2, or 15 � 15 μm2 scan size, then on 5 � 5 μm2,1.5� 1.5 μm2 and 1� 1 μm2 for a better resolution. The PEF andthen the PMswere layered and left to adsorb on amica substratefrom the same solutions as for the OWLS experiments. Profilo-metric section analysis allowed locating the PM patches on topof the PEF.

Fourier Transform Infrared Spectroscopy in Attenuated TotalReflection Mode. The structural changes of the adsorbed PEmultilayers and incorporated BR during the construction ofPEI-PSS-PAH-PM-PSS-PAH were followed by recordingthe FTIR-ATR spectra with a Bruker IFS 66V spectrophot-ometer. In these experiments, the PEFs were built on the ZnSecrystal of the fluid ATR cell, and the intensity of the evanescentwave propagating at the crystal/multilayer interface was mea-sured by a deuterated triglycide sulfate (DTGS) detector andaperture-type KBr, and cooled to the temperature of liquidnitrogen.

The multilayers were constructed in situ on the ATR crystalby successive adsorption of PEs diluted in the same buffer:25 mM MES, 25 mM TRIS, 100 mM NaCl, pH 7.4. As H2Opresents a strong absorption in the infrared range also charac-teristic of proteins, D2O was used instead in all dilutions. ThePEs and the PM suspension were circulated with a peristalticpump above the ZnSe crystal (in a closed circuit) at a 0.5mL/minflow rate until the adsorbed amount reached saturation. Aftereach adsorption, the cell was emptied and rinsed by the buffersolution for about 10 min, thus the multilayers were constructedin the same way as in OWLS. A spectrum resulting from theaccumulation of 100 interferograms was acquired after eachadsorption stage with a resolution of 2 cm-1. The bufferspectrum was taken as reference.

Results and Discussion

Adsorption Kinetics of the PE/Protein LayersMonitored

In Situ by OWLS. The adsorption of PM at 0.15 M ionicstrength was performed on positively charged films ending withPAH, and negatively charged films ending with PSS. The twocorresponding types of construction were PEI-(PSS-PAH)2-PMxn-PAH-PSS-PAH and PEI-(PSS-PAH)2-PSS-PMxn-PSS-PAH-PSS (where n = 1-4, the number ofadsorption steps). The effect of the membrane concentrationon the construction of the PEF/PM matrix was also studied.

The recorded changes in the effective refractive index of thetransverse electric mode (NTE) versus time indicate the step bystep buildup of the PEI-(PSS-PAH)2 and the PEI-(PSS-PAH)2-PSS films (Figure 1, Ia and IIa, respectively) onto theplanar waveguide and the following four-step adsorption of thePM at 30 and 150 μM protein concentration. Buildup wascontinued by adsorbing new PE layers on the top of the proteinlayer. The variation of the thickness of the adsorbed layers, ascalculated from the NTE and NTM values, during multilayerconstruction is presented in Figure 1Ib,IIb. In these curves,

adsorption of PMs becomes more visible than in the measuredNTE and NTM raw data, where the differences in the refractiveindices (nc) of the PM suspension compared to that of the buffer(see Supporting Information) obscure the signal of adsorption.However, the insets presenting a zoom of the NTE raw dataduring the adsorption and rinsing steps of the PM reveal thesignal variation due to the differences in the refractive indices ofthe PM suspension and the buffer.

If we compare the adsorption of the PM on PAH and PSSending PEFs, it is clear that adsorption of PM is possible on boththe positively and negatively charged PEs, even if there aredifferences. Adsorption on the negatively charged PSS is rathersurprising, as the surface of the PM bears mostly negativecharges (isoelectric point pI = 5.2).17,26 This behavior rendersit likely that other than electrostatic phenomena are also con-tributing to the adsorption process of the PMs on chargedsurfaces. At first sight, adsorption of PM exhibits relatively fastkinetics, as saturation is reached within 10-15 min. However,we have to note here that, when rinsing was performed imme-diately, protein was removed (data not shown). A number ofexperiments like those in Figure 1 let us conclude that anadsorption time of about 1 h is needed to obtain an irreversiblebinding of PM, presumably due to a second process of rearran-gement of the PMs and/or the PEF surface. Moreover, whenadsorbed on a negatively or positively charged surface, the PMscan be incorporated only by new bilayers starting by theoppositely charged PEFs. PEF layering onto the PM waspossible only in this way, and no stable adsorption of PE ontothe PM layer was observed when layering was continued withidentically charged PEs: PSS-PM-PSS, or PAH-PM-PAH.This is demonstrated by the full removal of the layer of likecharge during rinsing, but buildup of a new layer, mostlyresisting rinsing from the oppositely charged PE (Figure 1, Iband IIb).

The layer-by-layer increase in the thickness (Figure 1, Ib andIIb) of the obtained architectures shows that, at low concentra-tion (30 μM) PM interacts strongly with the positively terminat-ing (PAH) PEF and a second injection of 150 μM concentratedPM contributes with an additional protein layer. On a 26.5( 1.5nm thick positively charged PAH-ending film, the PM30 wasforming a 3.5 ( 0.25 nm thick layer that grew to 11 ( 1.5 nmwhen a more concentrated solution of PM150 was added(Figure 1 Ib).When injected on a 29.5( 1.5 nm thick, negativelycharged PSS-ending film, the PM30 did not adsorb, and a secondinjection of concentrated PM150 was needed to form a layer of4.5 ( 0.5 nm thickness. Nevertheless, when more BR injectionswere carried out, no further protein adsorption was observed.Given that the PMhas a dimension of 500 nm� 5 nm (diameter�height),15,17 our measurements suggest that the PM formed adouble-layer on the positively charged PEF, whereas on thenegatively charged PEF a single-layer of PMhas been deposited.As to the driving forces of this two-step adsorption process ofthe PM, one has to consider both its net negative electric chargeand its strong permanent electric dipole moment perpendicularto the membrane plane (2.9 � 106 D at pH > 5).17,27 Thenegative charge plays an obvious role in promoting adsorptionon the positively charged PEF. The fact that PM adsorption wasalso observed on negatively charged PEF indicates that thermo-dynamic contributions (presumably entropic factors due tohydration of PM and PEF) can override the electrostatic

(26) Ross, P. E.; Helgerson, S. L.; Miercke, L. J.W.; Dratz, E. A. Biochim.Biophys. Acta 1989, 991, 134–140.

(27) Keszthelyi, L. Biochim. Biophys. Acta 1980, 598, 429–436.

DOI: 10.1021/la9002274Langmuir 2009, 25(9), 5159–5167 5161

ArticleSaab et al.

repulsion. The multilayer buildup after PM adsorption couldonly be continued with a layer of opposite charge, which is astrong indication of the oriented adsorption of PM as a result ofits permanent dipole moment.

To evidence the contribution of electrostatic interactions inthe multilayer buildup, experiments at three different ionicstrengths (0.05, 0.15, 0.55 M) were carried out. Figure 2 gathersthe results obtained when layering PMon a PEF ending with thepositively charged PAH. We found, by increasing the concen-tration of NaCl in the buffer, that the thickness of PEF increasesshowing that the dominant driving forces of these multilayerconstructions of PEs are electrostatic forces. As the content ofions in the solution increases, the charges of PEs are screened,thus they build in vermicular structures resulting in largethicknesses.11 To the contrary, when the ionic strength is low,PEs use all their charges to adhere to the surface, resulting inthinner layers.

The capacity of certain proteins to adsorb on either positivelyor negatively charged PE-terminated films has been previouslydescribed.9-12 In these works, the thickness of the protein layerand the amount of the uptaken protein varied largely with theionic strength and with the charge difference between theadsorbed protein and the terminating film layer, pointing outthe electrostatic origin of the interactions governing adsorption.

Contrary to these works, we found that the total thickness ofPM adsorbed on the positively (PAH) terminating multilayer

film is the same (10.0 ( 0.2 nm) for the three different ionicstrengths. However, the presence of the PM layers does not alterthe further, electrostatically driven, buildup of a new PSS-PAH

Figure 1. Changes of the effective refractive index of the transverse electric mode (NTE) upon buildup of PEI-(PSS-PAH)2-PM30-(PM150)x3-PAH-PSS-PAH and PEI-(PSS-PAH)2-PSS-PM30-(PM150)x3-PSS-PAH-PSS architectures (Ia and IIa, respectively)and the calculated film thickness (Ib, IIb). The PEI, PSS, and PAH PEs were adsorbed from a 5 mg/mL solution in 25 mM TRIS, 25 mMMES, and 100 mMNaCl (pH 7.4). PM was adsorbed from 30 and 150 μM solutions in the same buffer.

Figure 2. Comparison of layer-by-layer growth of the thicknessof the PEI-(PSS-PAH)2-PM-(PSS-PAH) or PEI-(PSS-PAH)2-PM-(PAH-PSS) architectures at different ionicstrengths as a function of the number of layers. The buffer was25mMMES, 25mMTRISwith 0, 100, and 500mMNaCl, pH7.4.

DOI: 10.1021/la9002274 Langmuir 2009, 25(9),5159–51675162

Article Saab et al.

bilayer in the sense that the thickness of the new layers dependssimilarly on the ionic strength as that of the PEF below thePM layer.

The construction of the multilayered PE/protein assemblymay also be affected by cooperative phenomena depending onthe concentration of the PM suspension in contact with theadsorption surface. Thus experiments were performed when PMwas adsorbed onto the PEF in one or two steps at differentconcentrations. The resulting PM layer thicknesses (averages ofthree experiments per point) as a function of ionic strength andprotein concentration are gathered in Figure 3.

The thickness of the PM layer adsorbed on a positively (PAH)terminating film at a low PM concentration of 30 μM dependson the ionic strength: it is very small at a low concentration ofNaCl, it passes through a maximum at the ionic strength of0.15 M, and then decreases again at high, 0.55 M salt concen-trations. This points out the electrostatic character of theinteractions between the PEF and PM at low membrane con-centration.28 Adsorbed at this low concentration and from a0.15M buffer, the obtained thickness of the PMwas∼3 nm on apositively (PAH) terminating film, suggesting an incompletesurface coverage. A second injection after rinsing at the samelow concentration (PM30PM30 on PAH) contributes with somemore adsorbed material, resulting in a 5nm thick PM layer thatcorresponds to a monolayer of 5 nm high PMs. When a secondinjection of more concentrated (150 μM) PM was carried out,the obtained thickness increased up to 11 nm. This correspondsto the formation of a bilayer of PMs. The formation of a secondlayer of PMwhen adsorbed on a positively terminating film wasobserved for all the ionic strengths. The independence of theobtained layer thickness on the ionic strength demonstrates that,at high PM concentration, other driving forces than just electro-static ones contribute to the adsorption process. Moreover, thissecond class of interactions seems to promote the adsorption oftwo layers. The layering process saturates with the formation ofa bilayer of PMs, since when 150 μM concentrated PM wasadsorbed in one, or two steps, the same thickness of about 9 nmwas obtained. As previously noted, a third and a fourth protein

injection resulted in no further material adsorption. The thick-ness of the first PM layer deposited from the 30 μM suspensionat 150mM ionic strength on top of the PSS (negative) upper PEFsurface was measured as ∼1 nm, and the total thickness afterrinsing and additional deposition from the 150 μM PM suspen-sion became ∼5 nm. The latter corresponds to a monolayer ofPM on the PSS ending PEF.

The calculated refractive indices of adsorbed PEF-PM layerscan provide information on the hydration state of the obtainedmatrix, therefore we plotted them as a function of ionic strengthand PM concentration (Figure 4).

We can note that the refractive index of the adsorbed PEF-PM layer decreases with increasing ionic strength: in the pre-sence of ions (0.55 M), the obtained coating seems to have amore hydrated structure than at low ionic strengths (0.05 M),and this effect was already observed for other systems, too.27

A slight decrease in the refractive indices (n) with the ionicstrength of thinner adsorbed PE layers has been reportedpreviosuly.11 Our results indicate a more accentuated decreasein n that might be related to the fact that the thicker multilayeredfilms obtained in our work can get more easily hydrated. Whencomparing the refractive indices of the obtainedmultilayers withPM adsorbed on positively (PAH) or negatively (PSS) terminat-ing film (not shown), the same values were obtained. Thus theadsorbed PM cover layer does not influence the optical proper-ties of the multilayer structure. This might be important forfurther optoelectronic applications of these combined PE/BRarchitectures.

Thus adsorption of PMs is a complex process depending onthe membrane concentration and surface charge and can beinterpreted as follows: (i) for low PM concentrations, as timeevolves, the PAH PE readjusts its conformation leading to atighter interaction with PM that prevents further adsorption;(ii) for high PM concentrations, there is no time for such areadjustment and PE loops can emerge out of the first adsorbedprotein layer, providing new binding sites for a second subse-quent PM adsorption. The layering process is limited by theinterpenetration depth of the PEs, in our case, at two layers ofPM. The same effects were previously observed for otherproteins adsorbed on the same type of PEF.28 However, in thecase of highly concentrated PM, adsorption of a second layermust be promoted by other types of interactions (hydrophobic,

Figure 3. Thickness of the PM layer adsorbed on PAH-endingPEFs as a function of the ionic strength of the buffer and proteinconcentration. Experimental conditions were the same as forFigure 2.

Figure 4. The calculated refractive indices of the adsorbed PEF-PM as a function of the ionic strength and membrane concentra-tion. Experimental conditionswere the same as for Figures 2 and 3.

(28) Ladam,G.; Schaaf, P.; Decher, G.; Voegel, J.-C.; Cuisinier, F. J. G.Biomol.Eng. 2002, 19, 273–280.

DOI: 10.1021/la9002274Langmuir 2009, 25(9), 5159–5167 5163

ArticleSaab et al.

effect of hydration) to override the electrostatic repulsionbetween deposited PMs. This process dominates at highPM concentrations and only on positively charged (PAH-terminating) surface. To the contrary, on negatively charged(PSS ending) film adsorption never exceeds the formationof a single layer of PM, probably because of the dominationof the repulsive forces between the negatively chargedPSS and PM. A schematic representation of the proposedadsorption process of the PM patches onto the PEFs in anoptimal condition of medium ionic strength is illustrated inFigure 5.

Moreover, in the two cases, the orientation of the depositedPMs is expected to be different. It is known that in neutral andalkaline environments, the cytoplasmic side of PM fragmentsbears more negative charges than the extracellular side, and thepermanent electric dipole moment, which is parallel to themembrane normal, points toward the extracellular side.17 Whenadsorbed on the positive PAH, PMs should preferably face withtheir cytoplasmic side presenting more negative charges towardPAH. The second PM layer is then expected to take up the sameorientation as a result of the interaction of the dipole moment ofthe approaching PMs with the electric field close to the PEsurface, to the electrostatic interactionwith the charges on PAH,and despite the electrostatic repulsion between the PMs. Thisshould result in a final PM layer facing up with the extracellularside presenting less negative and more positive charges for theadsorption of a new PSS layer. Contrarily, when PAH wasinjected, no adsorption was observed, indicating againthe extracellular orientation of the PM toward the bulk(Figure 1a,b). When adsorbed on the negatively charged PSS,PMs are expected to face it with their extracellular side present-ing less negative charges. Thus, finally, a PM layer facing upwiththe cytoplasmic side (strongly negative) is formed, allowingfurther adsorption of only the positive PAH and not of thenegative PSS, as it was indeed observed (Figure 1a,b). Hence, wemight conclude that adsorption resulted in the formation oforiented single or double layers of PMs, facing the bulk with thecytoplasmic or the extracellular side on a negatively or positivelycharged surface, respectively.Morphological Studies by AFM. The morphological study

of the PE/protein layers was performed by AFM leading tovaluable information on the surface of the obtained matrices.First, the images of the PAH- or PSS-terminating layers of PEF,before PM injection, were recorded in liquid phase and tappingmode, revealing a rather smooth surface of a roughness of 3 nmexpressed by root-mean-square. In Figure 6a, we show thesurface of the PAH-terminating layer, and the AFM image ofthe PSS layer was very similar (not shown). The deposition ofPM patches can then be noticed when PM at a concentration of150 μM was adsorbed in one step on the positive PAH surface(Figure 6b). The corresponding images for the PM adsorbed onPSS-terminating layers are shown in the Supporting Informa-tion. A surface coverage of 63% (Figure 6b) can be calculatedfor the PM fragments (encircled in red) adsorbed in one step onthe PAH-ending PEF. This value is around the theoreticaljamming limit of 54.6% predicted by the random sequential

adsorption (RSA) theory. 29 Themaximum surface coverage canslightly increase for polydisperse hard disk monolayers up to73%. 30 Thus we may conclude that the 63% surface coveragewith PMs is close to the formation of a monolayer. A secondexposure to the PM suspension promoted the adsorption of newPMs, and the surface became covered at 87% (Figure 6c), whichis substantially above the theoretical jamming limit. Acknowl-edging that the polydispersity of the PM suspension is the same,such a tight packing can only be explained if one considers that asecond layer of PMs is formed. Thus, our AFM images supportthe results obtained with OWLS, i.e., the formation of a bilayerof PM on the PAH-terminating PEF. The profilometric sections(in the Supporting Information) taken between different pointsof the surface clearly indicate a thickness of about 10 nm thatcorresponds to a double layer of PMs.

The OWLS experiments have also shown that the buildup ofPEF on the top of PM is possible. Thus, AFM images wererecorded to document this process as well. Indeed, the AFMimage presented in Figure 6d demonstrates an almost completecoverage of the PMs by a further bilayer of PSS-PAH.

When imaging at higher resolution, single PM patches can benoticed on the surface of both PEFs (Figure 7). They have atypical diameter of ∼1 μm as seen in the profilometric sections(see also Supporting Information). There are several previousstudies showing AFM images (in contact mode) with subnan-ometer resolution recorded on PMs adsorbed on freshly cleavedmica.31,32 Our AFM images, taken in tapping mode and with avery soft cantilever, were designed to monitor the arrangementof membrane patches adsorbed onto soft PE layers, thus nobetter resolution could be expected from these measurements.Secondary Structure of the Protein Monitored by FTIR-

ATR Measurements. To essay the secondary structure of BRduring PM adsorption, we performed FTIR spectroscopy inATR mode. The PEI-PSS-PAH-PM-PSS-PAH architec-ture was in situ built up on the ATR cell from the 0.15 M bufferbut diluted in D2O. Infrared spectra were recorded after eachadsorption step, thereby monitoring the layer-by-layer buildupof the PEF/PM architecture.

The appearance of three major characteristic bands of theprotein can be observed at 1665 cm-1,1545 cm-1, and 1455 cm-1

that correspond to the stretching vibrations of the CdO (amide Iband), the combination of bending modes of -N-H andstretching vibration of -C-N (amide II band), and to thescissors vibration of the CH2, respectively (Figure 8A). Thesmall peak around 1525 cm-1 arises from the ethylenic stretch-ing modes of the retinal chromophore in BR.33

The secondary structure of the membrane-bound BR, incor-porated into the PEF, was determined by fitting Lorentzian-shaped component bands to the 1700-1600 cm-1 region of the

Figure 5. Schematic representation of the adsorption process of the PMs on the top of the (a) PEI-(PSS-PAH)2 and (b) PEI-(PSS-PAH)2-PSS multilayered films. Positively charged PE loops can emerge out of the first adsorbed PM layer leading to subsequent PMadsorption.

(29) Senger, B.; Bafaluy, F. J.; Schaaf, P.; Schmitt, A.; Voegel, J.-C. Proc. Natl.Acad. Sci. U.S.A. 1992, 89, 9449–9453.

(30) Doty, R. C.; Bonnecaze, R. T.; Korgel, B. A.Phys.Rev. E. 2002, 65, 061503.(31) Muller, D. J.; Heymann, J. B.; Oesterhelt, F.; Moller, C.; Gaub, H.; Buldt,

G.; Engel, A. Biochim. Biophys. Acta 2000, 1460, 27–28.(32) Scheuring, S.; Muller, D. J.; Stahlberg, H.; Engel, H.-A.; Engel, A. Eur.

Biophys. J. 2002, 31, 172–178.(33) Marrero, H.; Rothschild, K. J. Biophys. J. 1987, 52, 629–635.

DOI: 10.1021/la9002274 Langmuir 2009, 25(9),5159–51675164

Article Saab et al.

FTIR spectra (Figure 8B). The amide I region is very complex: itcontains several overlapping component bands assignable todifferent secondary structure elements of the protein. Thenumber and the initial position of the component bands duringfitting were set to comport with literature data and with thepublished atomic resolution crystal structure of BR.16,34,35 Theresult of the fit to the final PSS-PAH-PM-PSS-PAH struc-ture is shown in Figure 8B .

Table 1 gathers the results of the deconvolution of the amide Iband, leading to a thorough structural analysis of BR whenadsorbed on a PAH-ending PEF, then covered with PSS, andfinally with PAH. The peak positions, amplitudes, and widthsremain unchanged within error when the PMs are incorporatedinto the PEF by depositing further layers on the PM layer.Moreover, the secondary structural compositions obtained inour experiments are in good agreement with literature data on

structural analysis based on amide I band fits.34,35 These fitsuniformly show a slightly different composition of secondarystructural elements than that obtained by atomic resolutionX-ray diffraction, notably an underestimation of the R-helicalcontent. However, compared to the already published resultsconcerning the secondary structure of BR in solution, our datareveal a slight decrease in the quantity of reversed turns in favorof a β-sheet for BR in adsorbed form, certainly due to theinteractions of these extra-membranous segments with thePEFs. The quantity of the most dominant structures as the Rhelices and disordered structure rests the same, thus we canconclude that the incorporation of PM into PEFdid not alter thestructure of membrane-bound BR.

An important observation of the infrared studies publishedon BR was that the overall frequencies of the amide I and IIvibrations for BR are at least 10 cm-1 higher than values foundfor most R-helical polypeptides and proteins.36 This has been

Figure 6. Surface topography of the (a) PEI-(PSS-PAH)2 multilayered film, (b) the PMs (PM150) adsorbed on the top of a PEI-(PSS-PAH)2multilayered film inone step, (c) and that obtained in two steps, as recordedbyAFMin liquid and tappingmode. (d)PMincorporationwas also monitored by imaging the surface of the PEI-(PSS-PAH)2(PM150)x2-PSS-PAH multilayer. PM membrane fragments areencircled in red. Experimental conditionswere the same as for the OWLSmeasurements; the protein was left to adsorb for 1 h froma 150 μMsuspension, then rinsed.

(34) Cladera, J.; Sabes, M.; Padros, E. Biochemistry 1992, 31, 12363–12368.(35) Cladera, J.; Torres, J.; Padros, E. Biophys. J. 1996, 70, 2882–2887. (36) Rothschild, K. J.; Clark, N. A. Biophys. J. 1979, 25, 473–488.

DOI: 10.1021/la9002274Langmuir 2009, 25(9), 5159–5167 5165

ArticleSaab et al.

explained by the presence of an RII helix34,35 and an unusualprimary sequence that promotes the specific interactions of sidegroups, thereby disrupting the normal R-helical conformation.

It was also concluded that an important condition for theappearance of the band at 1665 cm-1, and hence the RIIhelix, is the existence of interactions between monomers of BR

Figure 7. Surface topographyandprofilometric sectionsof thePMs (PM150) adsorbedon the topof (a) aPEI-(PSS/PAH)2multilayered filmand (b) aPEI-(PSS/PAH)2-PSSmultilayered film, as recordedbyAFMin liquid and tappingmode. Experimental conditionswere the sameas for the OWLS measurements.

Figure 8. (a) FTIR-ATR spectra monitoring the adsorption of consecutive layers of PEF starting with PEI-PSS, followed by PAH, PMPSS, and PAH. (b) Deconvolution of the amide I band of the membrane-bound BR incorporated into the PE multilayer. The experimentalconditions were the same as for the OWLS experiments.

Table 1. Secondary Structure Analysis of the PM Based on the Decomposition of the Amide I Region into Lorentzian-Shaped Component Bands

β-sheet disordered structures RI helix RII helix reversed turns

-PAH-PM 1638.2((2.0) 1649.5((3.2) 1655.9((1.0) 1665.2((0.5) 1685.0((1.5) Fa

17.97((4.2) 7.95((4.1) 6.26((1.5) 10.75((1.2) 14.92((1.8) Wb

28.32 8.13 12.00 45.73 5.82 %c

-PAH-PM-PSS 1637.8((2.6) 1649.7((4.0) 1656.1((1.3) 1665.3((0.5) 1685.3((2.0) F17.13((4.4) 8.85((8.4) 6.72((3.3) 10.82((1.4) 13.98((12.0) W27.37 9.62 11.82 45.88 5.32 %

-PAH-PM-PSS-PAH 1638.0((1.9) 1650.2((2.4) 1656.3((0.8) 1665.1((0.4) 1684.5((3.5) F18.14((5.9) 8.23((5.2) 5.88((2.1) 10.72((1.7) 15.31((21.5) W28.38 10.21 10.48 45.01 5.92 %

aF = vibration frequency in cm-1. bW = bandwidth. c% = percent of the deconvoluted band relative to the total amide I band intensity.

DOI: 10.1021/la9002274 Langmuir 2009, 25(9),5159–51675166

Article Saab et al.

forming trimers.37 These R-helical segments are oriented per-pendicularly to the membrane plane in either dry or hydratedfilms.38 Thus, the significant quantity (∼45%) of the RII-helicalform found in ourmeasurements suggests that the BRmoleculesincorporated into PEFs conserve their trimeric organizationwithin the PM patches. During buildup of PEF/PM matrix,vibrations of the methyl branched fatty acid chains originatingin phospholipids within the PM were also observed (see Sup-porting Information).38

To summarize, our FTIR results evidence the stability of thestructure of BR in adsorbed form and when incorporated withina PSS/PAH-type PE multilayer.

Conclusion

OWLS proved to be well suited to monitor in situ and in realtime the buildupof a combinedPE/membrane proteinmatrix: BRin its membrane bound form (PM)was adsorbed and successfullyincorporated into the PE multilayered films. The PMs form asingle or a double layer when adsorbed on a negatively orpositively charged surface, respectively. The formation of thicklayers of globular proteins extending to several times the greatestdimension of the protein has already been demonstrated, but thisis the first time that such observation is made for proteolipidmembranes. The driving forces of the adsorption process of thePM onto charged surfaces were evaluated by varying the ionicstrength of the solution as well as the membrane concentration.At high membrane concentration, rearrangement of PE loopscannot take place, presumably due to the kinetic competition byPM adsorption, and the remaining PE protrusions may promoteadsorption of a secondmembrane layer on the positively chargedPEF. This cannot be achieved on a negatively charged PEF, or atlower membrane concentration, where PE rearrangement mayprecede the encounter of further membrane patches from thesolution with the surface. PMs were successfully incorporatedinto the PE multilayers in a way that indicates that they areoriented on the top of the PEFs: when adsorbed on a positive or a

negative surface, they expose the extracellular or cytoplasmic sidetoward the bulk, respectively.

The AFM images revealed the morphology of the PMsadsorbed on either the positively or the negatively charged PEsurface.Minor structural changes of BRwhile adsorbed onto andincorporated within charged surfaces were observed by FTIRspectroscopy in ATRmode. The adsorption does not disturb thesecondary structure majority of R-helix and disordered structuresof the protein. The R-helix amount remains the same when newPE is adsorbed on the top of the BR layer, demonstrating the verystable structure of this protein in the PM, contrary to earlierobservations on adsorbed globular proteins, where β-sheet struc-tures were predominant.

Summarizing, charge-driven adsorption of PMs resulted in theformation of highly oriented layers facing the cytoplasmic orextracellular side toward the bulk. Surface charge, ionic strength,and protein concentration are parameters that can be used toproduce PE/membrane protein architectures with tunable thick-ness and hydration properties with a high local organization.Manipulating PMs in this waymight promote the development ofinteresting biomimetic materials for biophotonic, bioelectronic,or sensory devices.

Acknowledgment. This work was supported by thePhoremost European Network of Excellence, ProjectNo. 511616: “NanoPhotonics to Realise Molecular Scale Tech-nologies” and by the COST-EU Action MP0702: “Towardsfunctional sub-wavelength photonic structures”. L.Z. is thank-ful for the grant provided by the Languedoc-Roussillon Regionand for the Hungarian Scientific Research Fund (OTKAT049207).

Supporting Information Available: The measured refrac-tive indices of the solutions used in this study as a function ofionic strength and protein concentration; the surface topo-graphy and profilometric sections of the PMs adsorbed onthe top of the multilayered PEFs; the FTIR spectrum of thelast five layers of the PEF/PM structure in the range of 2800-3150 cm-1. This material is available free of charge via theInternet at http://pubs.acs.org.

(37) Torres, J.; Sepulcre, F.; Padros, E. Biochemistry 1995, 34, 16230–16326.(38) Jonas, R.; Koutalos, Y.; Ebrey, T. G. Photochem. Photobiol. 1990, 50,

1163–117.

DOI: 10.1021/la9002274Langmuir 2009, 25(9), 5159–5167 5167

ArticleSaab et al.