Buildup of Exponentially Growing Multilayer Polypeptide Films with

Institut Charles Sadron, Centre National de la Recherche Scientifique, Unité Propre 22, 6 rue Boussingault, 67083 Strasbourg Cedex, France, Institut ...
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Langmuir 2003, 19, 440-445

Buildup of Exponentially Growing Multilayer Polypeptide Films with Internal Secondary Structure Fouzia Boulmedais,† Vincent Ball,† Pascale Schwinte,‡ Benoit Frisch,§ Pierre Schaaf,† and Jean-Claude Voegel*,‡ Institut Charles Sadron, Centre National de la Recherche Scientifique, Unite´ Propre 22, 6 rue Boussingault, 67083 Strasbourg Cedex, France, Institut National de la Sante´ et de la Recherche Me´ dicale, Unite´ 424, 11 rue Humann, 67085 Strasbourg Cedex, France, and Laboratoire de Chimie Bioorganique, UMR 7514 CNRS-ULP, 74 route du Rhin, 67400 Illkirch, France Received August 22, 2002. In Final Form: October 17, 2002 The buildup and secondary structure of poly(L-glutamic acid)/poly(allylamine) (PGA/PAH) multilayer films were investigated by means of optical waveguide lightmode spectroscopy, quartz crystal microbalance, and Fourier transform infrared spectroscopy in attenuated total reflection mode. The thickness and the mass of these films grow exponentially with the number of deposited bilayers. Moreover, PGA undergoes a random/R-helix transition when interacting with PAH during the film buildup process. This structural transition leads to (PGA/PAH)i films with an R-helix content (contribution of the R-helices to the amide I band) that switches regularly between 30% and 40% during the film buildup, when the multilayer is alternatively brought into contact with the PAH and PGA solutions. The secondary structure of the film is thus entirely driven by the last deposited layer. The independence of the R-helix content with the number of deposited bilayers also strongly suggests that the film is structurally homogeneous over its whole thickness.

I. Introduction The alternate deposition of polyanions and polycations on charged surfaces became a versatile and easy way to coat surfaces with films of controlled architecture and thickness.1 These films are called polyelectrolyte multilayers. Since their discovery 10 years ago, many polyelectrolyte multilayers were constructed based on a large variety of polyanion/polycation couples, constituted of strong polyelectrolytes,2 weak polyelectrolytes,3 natural polyelectrolytes or synthetic polypeptides,4,5 and even proteins6 or charged particles.7 Depending upon the nature of the polyelectrolytes, the multilayers present layered “fuzzy” structures8 or are more ordered.9 The use of polypeptides can also lead to films with a high secondary structure analogous to those found for proteins. The interaction of poly(L-lysine) and poly(L-glutamic acid) leads for example to films presenting a high β-sheet content.10 * To whom correspondence should be addressed. † Institut Charles Sadron, Centre National de la Recherche Scientifique. ‡ Institut National de la Sante ´ et de la Recherche Me´dicale, Unite´ 424. § Laboratoire de Chimie Bioorganique, UMR 7514 CNRS-ULP. (1) Decher, G. Science 1997, 277, 1232. (2) Glinel, K.; Moussa, A.; Jonas, A. M.; Laschewsky, A. Langmuir 2002, 18, 1408. (3) Yoo, D.; Shiratori, S. S.; Rubner, M. F. Macromolecules 1998, 31, 4309. (4) (a) Picart, C.; Lavalle, Ph.; Hubert, P.; Cuisinier, F. J. G.; Decher, G.; Schaaf, P.; Voegel, J. C. Langmuir 2001, 17, 7414. (b) Lavalle, Ph.; Gergely, C.; Cuisinier, F. J. G.; Decher, G.; Schaaf, P.; Voegel, J. C.; Picart, C. Macromolecules 2002, 35, 4458. (5) Elbert, D. L.; Herbert, C. B.; Hubbell, J. A. Langmuir 1999, 15, 5355. (6) Lvov, Y.; Ariga, K.; Ichinose, I.; Kunitake, T. J. Am. Chem. Soc. 1995, 117, 6117. (7) Kotov, N. A.; De´ka´ny, I.; Fendler, J. H. J. Phys. Chem. 1995, 99, 13065. (8) Decher, G.; Schmitt, J. Prog. Colloid Polym. Sci. 1992, 89, 160. (9) Arys, X.; Laschewsky, A.; Jonas, A. M. Macromolecules 2001, 34, 3318. (10) Boulmedais, F.; Schwinte, P.; Gergely, C.; Voegel, J. C.; Schaaf, P. Langmuir 2002, 18, 4523.

This can be of interest for specific interactions with proteins or for applications in the biomineralization field.11 The aim of the present study is to investigate the secondary structure of poly(L-glutamic acid)/poly(allylamine) (PGA/PAH) multilayer films. PGA is known to undergo an R-helix to coil transition in solution by a pH increase. It will be shown that these films present a high R-helix content even at pH 7.4 where PGA should exist mainly in a random conformation.12 The contribution of the R-helices to the amide I band switches from 30 to 40%, independent of the number of bilayers, when the multilayered architecture is brought into contact alternatively with the PAH and PGA solutions. The whole secondary structure of the film thus entirely depends on the nature of the outer layer. II. Materials and Methods Polyelectrolyte Solutions. Polyelectrolytes of commercial origin were used for the buildup of the (PGA/PAH) polyelectrolyte architectures. PAH (cat no. 28,322-3, MW ) 50 000-65 000 g mol-1) was purchased from Aldrich, and PGA (P-4886, MW ) 50 300 g mol-1) was purchased from Sigma. All the polyelectrolytes were used without further purification at a 5 mg mL-1 concentration, prepared by direct dissolution of the adequate weights. Either Millipore filtered water (Milli Q-plus system) or deuterium oxide (99.9% D, Aldrich, cat no. 15,188-2) was used as the solvent. The polyelectrolytes were dissolved in filtered (Millex GV membranes, pore diameter of 0.22 µm) buffer, prepared from 2-(N-morpholino)-ethanesulfonic acid (MES, 25 mM, Sigma, M-8250), tris(hydroxy-methylaminomethane) (Tris, 25 mM, Gibco BRL, cat no. 15504-020), and sodium chloride (NaCl, 0.1 M). The buffer was adjusted at pH 7.40 ( 0.05 just before use. For all measurement techniques, the layer by layer (LbL) assembly was started onto negatively charged surfaces by adsorbing first positively charged poly(ethyleneimine) (PEI, Aldrich, cat no. 18,197-8, MW ) 750 000 g mol-1). (11) Falini, G.; Albeck, S.; Weiner, S.; Addadi, L. Science 1996, 271, 67. (12) Wada, A. Mol. Phys. 1960, 3, 409.

10.1021/la0264522 CCC: $25.00 © 2003 American Chemical Society Published on Web 12/21/2002

Buildup of Multilayer Polypeptide Films Optical Waveguide Lightmode Spectroscopy. Optical waveguide lightmode spectroscopy (OWLS) is an optical technique that allows one to determine the optical thickness and the refractive index of an adlayer deposited on a Si0.8Ti0.2O2 waveguide. The technique has been theoretically described elsewhere13 and experimentally applied to polyelectrolyte multilayers.14 A laser beam is directed on a diffraction grating imprinted in the waveguide. The incoupling angles for both transverse electric (TE) and transverse magnetic (TM) waves are then determined by changing the incident angle of the laser beam. To each incoupling angle corresponds an effective refractive index, NTE and NTM, respectively. The knowledge of the effective refractive indexes allows one to calculate the optical thickness and the refractive index of the film. The film deposited on the waveguide is sensed by an evanescent wave with a penetration depth typically of the order of 200 nm. When the film thickness exceeds largely this penetration length, the film behaves optically as an infinite medium. All experiments were performed on a home-built experimental setup13 with a He-Ne laser using ASI 2400 waveguides made of Si0.8Ti0.2O2 (Artificial Sensing Instruments, Zu¨rich, Switzerland). Each experiment was preceded by a cleaning procedure of the waveguides, first in a 2% (v/v) Hellmanex II solution (from HELLMA GMBH, Mu¨llheim, Germany) for 10 min followed by a 20 min rinse in 0.1 N HCl, both in a boiling water bath. This step was followed by an extensive water rinse. The waveguide was subsequently dried under a nitrogen stream, introduced in its holder, and connected to a three-hole sealed cover (injection port, buffer entrance port, and buffer exit port tubes). The measuring cell is tightly sealed to the chip by a circular perfluorinated O-ring (Kalrez, Dupont, Wilmington, DE) and has an internal volume of 37 µL. Once the cell was mounted, buffer was flushed through the cell at a constant flow rate (10.1 mL/h) with a syringe-pump. Measurements were started once constant values of the incoupling angles were reached (less than 10-5 absolute variation on the values of NTE and NTM). These values were used to calibrate the waveguide, namely, to calculate the optical parameters (nF, dF) of the oxide layer deposited on the supporting glass chip. The buildup of the polyelectrolyte multilayers was performed as follows. After the buffer flow was stopped, 100 µL of one of the polyelectrolyte solutions was manually injected in the cell through the injection port. The surface was then maintained in contact with the solution at rest. After 10 min of contact (sufficient to reach a constant value for NTE and NTM), the polyelectrolyte solution was replaced by a buffer solution which was injected in the cell under a constant flow rate of 10.1 mL h-1. The film was then left for 10 min in contact with the buffer (again sufficient to reach a constant signal). This was done alternatively with the polycation and the polyanion solution. These polycation/polyanion adsorption steps were repeated until the multilayer film was built to the desired number of layers. A film constituted by a PEI layer and i subsequent (PGA/PAH) bilayers will be denoted by (PGA/PAH)i and (PGA/PAH)i-PGA for PAH- and PGA-ending films, respectively. Quartz Crystal Microbalance. The quartz crystal microbalance (QCM-D) experiments were performed on a Q-Sense D 300 apparatus from Q-Sense AB (Gothenburg, Sweden) by monitoring the resonance frequencies of the silicium-coated crystal at four different frequencies (5, 15, 25, and 35 MHz). These data give, in principle, information about the adsorption process and the mechanical properties of the deposited film.15,16 In a first approximation, the frequency changes are proportional to the total mass constituting the film deposited on the crystal.17 However, this approximation holds in the case of homogeneous, nonslipping, and rigid films. At the beginning, the crystal was put into contact with a buffer solution for 10 min. The (PGA/PAH)i films were built allowing (13) Tiefenthaler, K.; Lukosz, W. J. Opt. Soc. Am. B 1989, 6, 209. (14) Picart, C.; Ladam, G.; Senger, B.; Voegel, J. C.; Schaaf, P.; Cuisinier, F. J. G.; Gergely, C. J. Chem. Phys. 2001, 115, 1086, 5796. (15) Voinova, M. V.; Rodahl, M.; Jonson, M.; Kasemo, B. Phys. Scr. 1999, 59, 391. (16) Ho¨o¨k, F.; Kasemo, B.; Nylander, T.; Fant, C.; Sott, K.; Elwing, H. Anal. Chem. 2001, 73, 5796. (17) Sauerbrey, G. Z. Phys. 1959, 155, 206.

Langmuir, Vol. 19, No. 2, 2003 441 alternate adsorption of the polyelectrolyte solutions on the crystal for 10 min at rest and rinse by buffer for 10 min. These durations were sufficient to reach constant values for the frequency changes and hence a constant amount of deposited polyelectrolyte. Fourier Transform Infrared Spectroscopy. The in situ Fourier transform infrared (FTIR) spectroscopy in attenuated total reflection (ATR) mode experiments were performed in deuterated MES-Tris-NaCl buffer at pD 7.4. D2O is used as the solvent instead of water because the amide I band of the polypeptide is affected by the strong water band absorption around 1643 cm-1 (O-H bending), whereas the corresponding vibration in D2O is found around 1209 cm-1.18 ATR infrared spectra were taken with an Equinox 55 FTIR spectrometer (Brucker, Germany), using a liquid-nitrogen-cooled MCT detector. The (PGA/PAH)i films were deposited on a ZnSe crystal. For the film buildup, the different solutions (about 3 mL) were flowed into an overhead in-compartment ATR cell (GRASEBY-SPECAC, U.K.), equipped with a top-plate (110 µL cell volume) fitted with a 45° trapezoidal ZnSe (internal reflection element) crystal (6 reflections; dimensions, 72 × 10 × 6 mm3). The solutions were introduced into the cell by a peristaltic pump at a flow rate of approximately 0.25 mL min-1. Alternate deposition of the polyanions and polycations was performed by flowing each polyelectrolyte solution for 12 min over the ZnSe crystal, followed by an 8 min buffer rinse. After each polyelectrolyte deposition, at the end of the rinsing step, single-channel spectra from 512 interferograms were recorded between 400 and 4000 cm-1 with 2 cm-1 resolution, using Blackman-Harris three-term apodization and the standard Bruker OPUS/IR software (version 3.0.4). Analysis of raw spectra obtained in the ATR mode was performed by subtracting from the PEI-(PGA/PAH)i or PEI-(PGA/PAH)iPGA spectra the contribution of the PEI spectrum. Moreover, all raw infrared spectra were Fourier smoothed according to a previously described procedure.19 The analysis was focused on the amide I band in the 1600-1700 cm-1 wavenumber range. This band was decomposed into individual components which correspond to different secondary structures of the polypeptide. The decomposition was performed using the SPSERV software (version 3.20, BCS Software: Dr. Csaba Bagyinka, Institute of Biophysics, Biological Research Center of the Hungarian Academy of Sciences, H-6701 Szeged, P.O. Box 521, Hungary). More details concerning spectral decompositions can be found in ref 19. The frequencies of the different components forming the amide I band were first determined by means of the second derivative of the Fourier smoothed spectrum. There was thus no arbitrary decomposition into a preset number of bands. Once the number of component bands was determined, the amide I band was fitted by using the components’ frequency, width, and intensity (proportional to the area under the peak) as fitting parameters. All component bands were assumed to be Gaussian. The correspondence of each component band with a given secondary structure was established by comparing the frequency of its maximum to the value given in the literature.20-23 We define the relative contribution of each component by the ratio of the area of each peak over the total area of the whole amide I band.

III. Results Multilayer Buildup. The (PGA/PAH)i film construction was followed by OWLS and QCM-D. Figure 1 shows the evolution of the effective refractive index NTE, measured by OWLS, during the buildup process. A similar qualitative evolution is obtained for NTM. While at the beginning the effective refractive index increases when the film is brought into contact with a new polyelectrolyte solution, the index becomes cyclic after the deposition of (18) Venyaminov, S. Y.; Prendergast, F. G. Anal. Biochem. 1997, 248, 234. (19) Schwinte, P.; Voegel, J. C.; Picart, C.; Haikel, Y.; Schaaf, P.; Szalontai, B. J. Phys. Chem. B 2001, 105, 11906. (20) Miyazawa, T.; Blout, E. R. J. Am. Chem. Soc. 1961, 23, 712. (21) Krimm, S. J. Mol. Biol. 1962, 4, 528. (22) Jackson, M.; Haris, P. I.; Chapman, D. Biochim. Biophys. Acta 1989, 998, 75. (23) Venyaminov, S. Y.; Kalnin, N. N. Biopolymers 1990, 30, 1259.

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Figure 1. Evolution of the effective refractive index NTE as obtained by OWLS during the alternate deposition of PGA and PAH layers on a precursor PEI layer. A line labeled A corresponds to the beginning of the injection of the polyanion, and a line labeled C corresponds to the beginning of the injection of the polycation. The inset shows the evolution of the refractive index of the multilayer film nA during the deposition of the seventh and eighth bilayers when “optical cycling” occurs; a line labeled R corresponds to the beginning of the rinsing step.

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Figure 3. Evolution of the normalized frequency (∆f/ν) as a function of the deposition time as obtained by QCM-D in the case of the (PGA/PAH) system: signal obtained from the 15 MHz (O), 25 MHz (b), and 35 MHz ([) harmonics. The inset shows the fit of the equation ∆f ) ∆f0 + ∆f1 exp(ibil/i1) to the experimental data (∆f/ν) obtained at 15 MHz at the end of the rinsing steps following each PAH deposition. The best fit is obtained for ∆f0, ∆f1, and i1 equal to 184 Hz, 147 Hz, and 0.44, respectively, at 15 MHz.

similar plot is obtained for the evolution of the optical mass of the film (nA - nc)dA versus the number of deposited layers, nA and nc representing the refractive index of the multilayer film and of the buffer, respectively. As shown in the inset of Figure 2, the thickness of (PGA/PAH)i films increases approximately in an exponential manner with the number of bilayers ibil:

dA ) dA,0 + dA,1 exp(ibil/i0)

Figure 2. Evolution of the optical thickness dA (nm), obtained by OWLS, as a function of the added polyelectrolyte layers for the (PGA/PAH) system. The inset shows the fit of eq 1 to the experimental data.

the sixth bilayer. Indeed, when this optical cycling occurs, each new PAH adsorption (respectively PGA) leads to a decrease (respectively increase) of NTE. The optical NTE and NTM signals can be analyzed by assuming that the film behaves as an homogeneous and isotropic monolayer characterized by a given thickness and a refractive index.14 However, only physically reasonable and reliable solutions are obtained for films having a thickness smaller than 200 nm which corresponds roughly to the penetration length of the evanescent wave (see the Appendix). The evolution of the film thickness dA with the number of deposited bilayers ibil is represented in Figure 2 for thicknesses ranging between 0 and 200 nm. A qualitatively

(1)

with values for dA,0, dA,1, and i0 equal to -18.7 nm, 17.4 nm, and 0.42, respectively. The fact that for ibil ) 0 a negative thickness is obtained by OWLS must be due to a smaller thickness of the first bilayer, the buildup process of this bilayer being different from that of the remaining film. Otherwise, we can notice that the contact of the film with a PGA solution leads to a smaller thickness increase as compared to that obtained from the subsequent contact of the film with the PAH solution. Similar results are observed by QCM-D (see Figure 3) where the normalized frequency shift (∆f/ν) is in first approximation proportional to the mass of the film. This normalized frequency shift (∆f/ν) corresponds to the different frequencies (15, 25, 35 MHz) during the film buildup, ν representing the overtone number of the frequency and 5 MHz being the fundamental frequency. During the whole film buildup, (∆f/ν) is almost independent of ν. This strongly indicates that the multilayer behaves as a rigid film and that the Sauerbrey relation constitutes a reasonable approximation to describe the data. The evolution of the signal is determined here up to the ninth bilayer. For films constituted of more than nine bilayers, the signal-to-noise ratio became so poor that no further measurements were possible. The evolution of (∆f/ν) after the deposition of six bilayers proves that the film continues to grow exponentially even when the optical signals evolve cyclically (see inset of Figure 3).

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Figure 4. Normalized infrared absorption spectra of (PGA/ PAH)4 (b) and (PGA/PAH)4-PGA (O) multilayers deposited on a PEI precursor film at pD 7.4 in deuterated Mes-Tris-NaCl buffer.

Multilayer Secondary Structure. PGA is known to undergo in solution random coil/R-helix transitions. Our main interest was thus to investigate the secondary structure of the (PGA/PAH)i multilayer film by FTIRATR. Attention was paid to the amide I band (1600-1700 cm-1) which originates from the stretching vibration of the carbonyl group of the peptide bonds of PGA. PAH does not display characteristic vibration bands in this wavelength range in IR spectroscopy. Typical spectra for (PGA/ PAH)4 and (PGA/PAH)4-PGA multilayers are given in Figure 4. The spectrum shifts toward smaller wavenumbers after contact of a (PGA/PAH)4 film with a PGA solution. To gain more insight into the structure of the film, the amide I band was further decomposed into individual component bands assigned to different secondary structures of the polypeptide. Three bands centered at 1627, 1643, and 1664 cm-1 were found. They were attributed to R-helices, random, and turn structures, respectively.20-23 Figure 5 shows the evolution of the relative contributions of different bands composing the amide I band of the (PGA/ PAH)i film during its buildup. The contribution of the R-helices will be called “R-helix content”. It remains constant and equal to 30% throughout the construction for PAH-ending films. When such films are put into contact with a PGA solution, the R-helix content increases. More precisely, after the deposition of the third bilayer, the R-helix content varies in a cyclic way between 30 ( 2% for (PGA/PAH)i films and 40 ( 2% for (PGA/PAH)i-PGA multilayers. The other two components also vary in a totally cyclic way. This suggests that after the deposition of the third bilayer, the multilayer becomes perfectly uniform. Moreover, the secondary structure of the whole film is fully driven by the outermost layer. IV. Discussion We found that (PGA/PAH)i films grow exponentially with the number of deposited bilayers. Two explanations are advanced in the literature to describe such a growth process: one is based on the diffusion “in” and “out” of the film of at least one of the polyelectrolytes constituting the multilayer,24 and the other is based on a charge mismatch25 (24) Picart, C.; Mutterer, J.; Richert, L.; Luo, Y.; Prestwich, G. D.; Schaaf, P.; Voegel, J. C.; Lavalle, P. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 12531.

Figure 5. Evolution of the relative contribution of the three components of the amide I band of PGA during the buildup of a (PGA/PAH)9 multilayer film: (a) R-helix, (b) random, and (c) turn contents.

between the polyanion and polycation which induces an increased roughness of the film. We will analyze our experimental results in the light of these proposed mechanisms. Let us first assume that the exponential growth is due to the diffusion of at least one of the polyelectrolytes (the polyanion for example) in and out of the whole film during the buildup steps. When the film is brought into contact with the polyanion solution, some polyanion chains diffuse into the film up to the substrate and others interact directly with the polycations present at the top of the film. This direct interaction of polyanions with polycations leads to a charge overcompensation, and the new negative excess charge at the top of the multilayer creates a negative potential energy barrier. During the following rinsing step of the film by pure buffer, part of the free polyanion chains present in the film diffuse out of it. This induces a decrease of the chemical potential of the free polyanion chains in the film and correlatively an increase of the energy barrier to be crossed by these chains to leave the film. The out diffusion process thus stops when this energy barrier becomes larger than kT. At the end of the rinsing step, free polyanion chains thus remain present in the film. When the multilayer is further brought into contact with the polycation solution, polycation chains interact with the polyanions present at the outer layer of the film. This leads to an excess charge reversal, the energy barrier disappears, and all the remaining free polyanions diffuse out of the film. When they reach the film/solution interface, these polyanion chains interact with the polycations present in the solution and form polyanion/polycation complexes which constitute the new outer layer of the film. When all the free polyanions have diffused out of the film, the buildup process stops. Because the film is in contact with a polycation solution, these chains will adsorb in excess at the outermost part of the film, creating a new positive charge excess. The film is then rinsed with buffer, (25) Bertrand, P.; Jonas, A.; Laschewsky, A.; Legras, R. Macromol. Rapid Commun. 2000, 21, 319.

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and a new bilayer deposition cycle can start again. At each cycle, such a deposition mechanism leads to the formation of a new polyanion/polycation outer layer whose mass is proportional to the amount of free polyanion chains present in the film before contact with the polycation solution. The mass of the new outer layer thus increases exponentially with the number of deposition cycles. The diffusion of the polyanions into the film is accompanied by a change of the refractive index even near the deposition substrate. This could explain the cyclic behavior of the effective refractive indexes measured by OWLS in conditions where the film becomes so thick that the evanescent wave senses only part of the multilayer near the substrate. The smaller increase of the mass of the film measured by QCM-D after each contact of the film with the PGA solution compared to that found after the subsequent contact with the PAH solution suggests that PGA chains predominantly diffuse within the film. Indeed, this diffusion in the film should be accompanied by water removal so that the total mass of the film could remain more or less constant, as measured by QCM-D. On the other hand, when such a film is brought into contact with the PAH solution, the PGA diffusion out of the film is accompanied by a novel entrance of water in the multilayer and by the addition of the new PGA/PAH complex outer layer. The validity of this model could already be proven for two exponentially growing systems: hyaluronic acid/poly(L-lysine)24 and poly(L-glutamic acid)/poly(L-lysine).4 In both cases, the OWLS and QCM-D signals evolved qualitatively in a very similar way as here for the present PGA/PAH system. Indeed, the frequency shifts measured by QCM-D increased exponentially with the number of deposited bilayers, the film thickness determined by OWLS increased similarly, and when large thicknesses (about 200 nm) were reached, the effective refractive indexes evolved in a similar cyclic way. Moreover, the in and out diffusion process of at least one of the polyelectrolytes through the whole film was directly visualized by confocal laser scanning microscopy for the hyaluronic acid/ poly(L-lysine)24 and poly(L-glutamic acid)/poly(L-lysine) system.26 Besides, the cyclic changes of the secondary structure of PGA (contribution of the R-helices to the amide I band) between two constant values over the entire film buildup can have at least two origins: either the free PGA chains within the film more easily form R-helices than the chains involved in the PGA/PAH complexes or the free PGA chains induce a decrease of the local pH in the film during its diffusion. Such a pH decrease certainly takes place because the charges carried out by the free chains must be compensated by small ions to have a film which remains neutral. This gives rise to a Donnan potential that increases the presence of cations, and thus also of protons, in the film. It is remarkable that under the film buildup conditions (buffered pH 7.4 solutions), PGA presents such a high R-helix content. This suggests that the apparent pH in the film is lower than in the contact solution. Such an effect was already observed in other multilayer systems.27 Finally, the fact that the R-helix content switches between two constant values over the whole film buildup process indicates that the secondary structure of the film is homogeneous over its entire thickness film and thus does not evolve with the distance from the substrate. The second explanation of the “exponential” growth of multilayer films reported in the literature is based on a charge mismatch between the polyanion and the poly(26) Jessel, N., et al. Manuscript in preparation. (27) Klitzing, R.; Mo¨hwald, H. Langmuir 1995, 11, 3554.

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cation constituting the film. This could lead to an increased film roughness and thus to a “superlinear” growth regime. In this case, when the film becomes thick, the cyclic behavior of the effective refractive index observed by OWLS could be attributed to a change in the film structure up to the film substrate, consecutive to a change of the nature of the outer layer. Such a structural change could be due to a pH change over the entire film which could also lead to the observed modifications of the PGA secondary structures. Long-range pH changes extending over several nanometers were already observed in linearly growing films.28 They were attributed to local electrostatic potential changes originating from the excess charge of the outer layer. However, it is difficult to imagine that such a potential decrease within the film due to ion screening could extend over more than 200-300 nm, even if the Debye length can become very large inside of the film due to a reduced concentration of small ions. Such a large extension is required for the secondary structure of the film to remain constant over the entire multilayer architecture. Moreover, one would also expect that charge mismatches between the two polyelectrolytes would not lead to dense films, so that small ions could easily diffuse within the film and thus reduce the Debye length, even if the charges of the polyanions would be perfectly compensated by those of the polycations. Finally, even if such a growth mechanism could lead to a superlinear increase of the mass of the film with the number of deposited bilayers, there is no reason for the increase to become truly exponential. Such an exponential behavior was clearly observed for the PGA/PAH system by two independent techniques. V. Conclusions In this paper, we investigated (PGA/PAH)i multilayered architectures. These films possess an internal secondary structure mainly composed by R-helices. The structure of these films switches between two states, depending upon the nature of the outer layer. The mass and thickness of these multilayers increase exponentially with the number of deposition cycles. Two explanations were proposed to explain the experimental observations: the first is based on the in and out diffusion of PGA molecules through the whole film during each buildup cycle, and the second on a film roughness increase during the buildup process accompanied by structural changes of the film induced by the outer nature of the layer. Whereas the second explanation seems difficult to justify for the PGA/PAH system, the diffusion-based mechanism can account naturally for all the observed features. Moreover, its validity has been proven experimentally for other exponentially growing films. Other multilayer films based on polypeptides will be investigated in the future to extend the present observations. It would be of particular interest to define films with buildup conditions allowing over a large range a fine secondary structure tuning and thus to mimic secondary protein structures. Such films could become interesting for specific protein interactions or the induction of calcium/phosphate or calcium/carbonate nucleation. Acknowledgment. The work was supported by the programs Action Concerte´e Incitative “Technologie pour la sante´” and “Surfaces interfaces et conception de nouveaux mate´riaux” from the Ministe`re Franc¸ ais de la (28) Feng Xie, A.; Granick, S. Macromolecules 2002, 35, 1805.

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Recherche and by the Centre National de la Recherche Scientifique program “Physique et chimie du vivant”. One of the authors, P. Schwinte´, thanks the Faculty of Odontology of Strasbourg for financial support. Appendix The interpretation of the cyclic evolution of the effective refractive indexes determined by OWLS for thick (PGA/ PAH)i films is based on the fact that OWLS is only sensitive to regions extending roughly up to 200-300 nm from the substrate. This was verified by following the buildup process of a linearly growing film: poly(styrene sulfonate)/ poly(allylamine) (PSS/PAH)n. For this film, it is also expected that none of the polyelectrolyte diffuses through the multilayer so that its local structure near the substrate should remain unchanged when additional (PSS/PAH) bilayers are added on the top of the film.29 Figure 6 shows the evolution of NTE and of the film thickness over the entire buildup process. As expected, the film thickness increases linearly with the number of deposited bilayers. For film thicknesses of about 200 nm, the effective refractive indexes saturate. No further changes of NTE and NTM are observed for the (PSS/PAH)i films once the leveling off step is reached, contrary to what is found for the exponentially growing films. This shows that the penetration length of the evanescent wave sensing the film in OWLS is of the order of 200 nm. In this case, once the optical signal saturated, negative values for the optical (29) Lo¨sche, M.; Schmitt, J.; Decher, G.; Bouwmann, W. G.; Kjaer, K. Macromolecules 1998, 31, 8893.

Figure 6. Evolution of NTE with ibil, the number of deposited PSS/PAH bilayers for the (PSS/PAH) system. The inset shows the evolution of the optical thickness dA as a function of ibil. The polyelectrolytes were solubilized at 5 mg mL-1 in filtered 10 mM Tris in the presence of 0.1 M NaCl at pH 7.4.

thickness were obtained by analyses of the optical data by the homogeneous and isotropic monolayer model. LA0264522