Polyelectrolytes

After deposition, the cellulose component occurred as a double layer with .... transform infrared since OH and OD stretching bands are separated by ab...
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Langmuir 2008, 24, 3452-3458

Structural Details of Cellulose Nanocrystals/Polyelectrolytes Multilayers Probed by Neutron Reflectivity and AFM Bruno Jean,*,† Fre´de´ric Dubreuil,† Laurent Heux,† and Fabrice Cousin‡ Centre de Recherche sur les Macromole´ cules Ve´ ge´ tales (CERMAV-CNRS)§, BP 53, 38041 Grenoble Cedex 9, France, and Laboratoire Le´ on Brillouin CEA-CNRS, Saclay 91191 Gif-sur-YVette Cedex, France ReceiVed October 2, 2007. In Final Form: January 10, 2008 Neutron reflectivity measurements and AFM observations were used as complementary techniques to investigate multilayered films consisting of alternating sheets of rigid cellulose nanocrystals and flexible poly(allylamine hydrochloride) (PAH) prepared by the layer-by-layer assembly technique. Both techniques showed that smooth films with a high load of cellulose could be obtained. After deposition, the cellulose component occurred as a double layer with different densities: 50% and 25% for the lower and upper layer, respectively. A linear growth of the multilayer and the presence of a Bragg peak on neutron reflectivity curves indicated the formation of a well-ordered structure resulting from entropy-driven assembly and smoothening effect of the flexible PAH macromolecules. The possible alignment of the nanocrystals when anisotropic suspensions were used is also shown and opens the route to an improved control of the architecture of these multilayers.

Introduction The processing of high-performance materials from nanoparticles is currently attracting fundamental and industrial interest. In this context, nanoelements presenting high aspect ratio, such as carbon nanotubes (CNTs) or clays platelets, have received significant attention due to their unique mechanical, electrical or transport barrier properties. In particular, it was shown that these particles could be used for the preparation of nanocomposites with enhanced characteristics.1-3 To meet the recent environmental awareness and new economic standards, nanomaterials based on natural and renewable resources are gaining substantial interest. For such products, cellulose nanocrystals (CNXLs) appear as very good candidates, since they offer a quite unique combination of high physical properties and environmental friendly characteristics. CNXLs are commonly obtained by sulfuric acid hydrolysis of native cellulose leading to stable aqueous suspensions of rodlike nanocrystals whose size and aspect ratio depend on their biological origin.4,5 These nanorods exhibit spectacular mechanical properties with an elastic modulus, E, in the range 120-145 GPa6-8 comparable to that of aramid fibers (Kevlar 149, 156 GPa) and only 2-8 times lower than that of * To whom correspondence should be addressed. E-mail: bruno.jean@ cermav.cnrs.fr. † Centre de Recherche sur les Macromole ´ cules Ve´ge´tales. ‡ Laboratoire Le ´ on Brillouin CEA-CNRS. § Affiliated with Universite ´ Joseph Fourier and member of Institut de Chimie Mole´culaire de Grenoble. (1) Baughman, R. H.; Zakhidov, A. A.; de Heer, W. A. Science 2002, 297, 787-792. (2) Powell, C. E.; Beall, G. W. Curr. Opin. Solid State Mater. Sci. 2006, 10, 73-80. (3) Salvetat, J. P.; Bonard, J. M.; Thomson, N. H.; Kulik, A. J.; Forro, L.; Benoit, W.; Zuppiroli, L. Appl. Phys. A 1999, 69, 255-260. (4) Dong, X. M.; Kimura, T.; Revol, J. F.; Gray, D. G. Langmuir 1996, 12, 2076-2082. (5) Revol, J. F.; Bradford, H.; Giasson, J.; Marchessault, R. H.; Gray, D. G. Int. J. Biol. Macromol. 1992, 14, 170-172. (6) Matsuo, M.; Sawatari, C.; Iwai, Y.; Ozaki, F. Macromolecules 1990, 23, 3266-3275. (7) Nishino, T.; Takano, K.; Nakamae, K. J. Polym. Sci. Part B: Polym. Phys. 1995, 33, 1647-1651. (8) Sturcova, A.; Davies, G. R.; Eichhorn, S. J. Biomacromolecules 2005, 6, 1055-1061.

single-walled CNTs (300-1000 GPa).9 Moreover, cellulose, the most abundant polymer on earth, benefits from low cost, biodegradability, biocompatibility, low density, and relative thermal stability. Over the past 15 years these features have motivated studies on nanocomposite materials using cellulose nanocrystals as fillers, thus mimicking natural composites. It was shown that a drastic improvement in storage modulus above the glass transition temperature (Tg) could be obtained for films of synthetic polymer lattices when a few percent of tunicin (an animal cellulose) nanocrystals had been added.9,10 Methods to control the orientation of the nanorods have also been developed at the same time. Alignment of cellulose and chitin crystallites perpendicular to static magnetic field has been demonstrated.11 More recently, Kimura et al. have successfully achieved an uniaxial orientation of cellulose nanocrocrystals using rotating magnetic fields.12 For crystal structure analysis, methods based on shear13 or elongational flow were used to prepare specimens of highly oriented cellulose nanocrystals. Initially, the orientation of CNXLs was restricted to aqueous suspension, but more recently, thanks to adequate surface chemistry, nonflocculated CNXLs suspensions could also be obtained in apolar solvents. These suspensions could be oriented by an electric field to prepare textured materials.14 Potential applications of CNXLs could also arise from the preparation of thin films with controlled architectures giving rise to tunable optical or mechanical properties. Layer-by-layer (LbL) assembly is a simple and versatile method to build tailor-made multilayered films with a high loading of materials.15,16 The technique consists in a sequential adsorption on a substrate (e.g., a cleaned silicon wafer or a glass slide) of oppositely charged compounds. First developed (9) Favier, V.; Chanzy, H.; Cavaille, J. Y. Macromolecules 1995, 28, 63656367. (10) Samir, M. A. S. A.; Alloin, F.; Dufresne, A. Biomacromolecules 2005, 6, 612-626. (11) Sugiyama, J.; Chanzy, H.; Maret, G. Macromolecules 1992, 25, 42324234. (12) Kimura, F.; Kimura, T.; Tamura, M.; Hirai, A.; Ikuno, M.; Horii, F. Langmuir 2005, 21, 2034-2037. (13) Yoshiharu, N.; Shigenori, K.; Masahisa, W.; Takeshi, O. Macromolecules 1997, 30, 6395-6397. (14) Bordel, D.; Putaux, J. L.; Heux, L. Langmuir 2006, 22, 4899-4901. (15) Decher, G. Science 1997, 277, 1232-1237. (16) Decher, G.; Hong, J. D.; Schmitt, J. Thin Solid Films 1992, 210, 831835.

10.1021/la703045f CCC: $40.75 © 2008 American Chemical Society Published on Web 03/07/2008

Structural Details of Multilayers

for polyelectrolytes, the method has since been expanded to include various nanobuilding blocks such as clays, proteins, or carbon nanotubes.17-20 LbL assembly thus provides a useful tool to prepare structured thin functional films incorporating nanoparticles with numerous potential applications ranging from mechanical and chemical sensors21 to biological materials,22,23 smart containers for drug delivery,24 optical materials,25,26 and superhydrophobic coatings.27 LbL assembly has already been applied to the preparation of multilayered films based on cellulose nanocrystals.28-32 The feasibility of LbL assembly of short and low aspect ratio CNXLs from cotton was demonstrated with polycations, namely poly(allylamine hydrochloride) (PAH)28 and poly(diallyldimethyl ammonium chloride) (PDDA).29 Working with cellulose and PAH, Cranston and Gray have performed a more detailed morphological and optical characterization of such multilayered films prepared by solution dipping and spin coating.31 In the latter case, dense, thick, and radially oriented films were observed. Spin coating has also been used by Kontturi et al. who recently reported on the adsorption of CNXLs on cationic titania and anionic silica surfaces by spin coating from dilute suspensions.33 AFM revealed that small aggregates were formed on silica whereas a uniform bidimensional distribution was observed on titania. This morphological difference is attributed to the electrostatic adsorption of CNXLs on positively charged titania surface in addition to the spin coating deposition. More recently, Podsiadlo et al. have used tunicin CNXLs presenting a substantially high aspect ratio. Using these nanocrystals, these authors were able to build LbL films with antireflective properties resulting from a highly porous architecture.30 Despite these achievements, a precise description of the structural details of these multilayered samples remains to be done. In this paper we present a detailed structural investigation of films based on the multilayer assembly of cotton CNXLs and PAH. For the characterization of these products, we have combined the advantage of two complementary techniques, namely neutron reflectivity and atomic force microscopy (AFM), thus yielding structural information both in direct and reciprocal space. Neutron reflectivity, which has already been applied to the characterization of other polyelectrolyte multilayers systems,34,35 provides structural details in the direction perpendicular (17) Kleinfeld, E. R.; Ferguson, G. S. Science 1994, 265, 370-373. (18) Caruso, F.; Caruso, R. A.; Mohwald, H. Science 1998, 282, 1111-1114. (19) Lvov, Y.; Ariga, K.; Ichinose, I.; Kunitake, T. J. Am. Chem. Soc. 1995, 117, 6117-6123. (20) Rouse, J. H.; Lillehei, P. T. Nano Lett. 2003, 3, 59-62. (21) Loh, K. J.; Kim, J.; Lynch, J. P.; Kam, N. W. S.; Kotov, N. A. Smart Mater. Struct. 2007, 16, 429-438. (22) Chluba, J.; Voegel, J. C.; Decher, G.; Erbacher, P.; Schaaf, P.; Ogier, J. Biomacromolecules 2001, 2, 800-805. (23) Richert, L.; Lavalle, P.; Vautier, D.; Senger, B.; Stoltz, J. F.; Schaaf, P.; Voegel, J. C.; Picart, C. Biomacromolecules 2002, 3, 1170-1178. (24) Sukhishvili, S. A. Curr. Opin. Colloid Interface Sci. 2005, 10, 37-44. (25) Nolte, A. J.; Rubner, M. F.; Cohen, R. E. Langmuir 2004, 20, 33043310. (26) Hattori, H. AdV. Mater. 2001, 13, 51. (27) Zhai, L.; Cebeci, F. C.; Cohen, R. E.; Rubner, M. F. Nano Lett. 2004, 4, 1349-1353. (28) Cranston, E. D.; Gray, D. G.; Barrett, C. J. Abstracts, 32nd Northeast Regional Meeting of the American Chemical Society, Rochester, NY, United States, October 31-NoVember 3 2004, GEN-332. (29) Podsiadlo, P.; Choi, S.-Y.; Shim, B.; Lee, J.; Cuddihy, M.; Kotov, N. A. Biomacromolecules 2005, 6, 2914-2918. (30) Podsiadlo, P.; Sui, L.; Elkasabi, Y.; Burgardt, P.; Lee, J.; Miryala, A.; Kusumaatmaja, W.; Carman, M. R.; Shtein, M.; Kieffer, J.; Lahann, J.; Kotov, N. A. Langmuir 2007, 23, 7901-7906. (31) Cranston, E. D.; Gray, D. G. Biomacromolecules 2006, 7, 2522-2530. (32) Cranston, E. D.; Gray, D. G. Sci. Technol AdV. Mat. 2006, 7, 319-321. (33) Kontturi, E.; Johansson, L. S.; Kontturi, K. S.; Ahonen, P.; Thune, P. C.; Laine, J. Langmuir 2007, 23, 9674-9680. (34) Schmitt, J.; Grunewald, T.; Decher, G.; Pershan, P. S.; Kjaer, K.; Losche, M. Macromolecules 1993, 26, 7058-7063.

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to the surface, including thickness, density, and roughness, whereas AFM is adapted to observe the surface topology. Experimental Section Materials. Polycations poly(ethylene imine) (PEI, Mw ) ∼25 000) and poly(allylamine hydrochloride) (PAH, Mw ) ∼70 000), the polyanion poly(sodium 4-styrene sulfonate) (PSS, Mw ) ∼70 000), and other chemicals were purchased from Sigma-Aldrich and used as received. Cotton linters were provided by Rhoˆne-Poulenc Tubize Plastics (Belgium) and used as the cellulose source without any further purification. Suspensions of Cellulose Nanocrystals. Nanocrystals suspensions were prepared of either deuterated or hydrogenated cellulose by the acid hydrolysis described further below. The deuteration step was achieved using the hydrothermal intracrystalline deuteration method described by Nishiyama et al.36 as follows. Cotton linters (15 g) were dispersed in a 0.1 N NaOD/D2O solution and placed in an autoclave for 1 h at 210 °C, resulting in the replacement of intracrystalline OHs by OD groups. This deuteration could be easily monitored by Fourier transform infrared since OH and OD stretching bands are separated by about 1000 cm-1. An important point of this treatment is that the cellulose crystallinity is left unaffected. Once redispersed in H2O, OD groups at the crystal surface will be turned into OH but OD groups in the crystalline part will be maintained. Deuterated and unmodified cotton linters were hydrolyzed according to the method described by Revol et al.5 by treatment with 65% sulfuric acid during 30 min at 63 °C. The suspensions were washed by centrifugation, dialyzed against distilled water until neutrality, and ultrasonicated for 4 min with a Branson B-12 Sonifier equipped with a microtip. After these treatments, the suspensions were filtered through 8 µm and then 1 µm cellulose nitrate membranes (Sartorius) and stored with mixed bed resin (Sigma tmd-8) in order to eliminate residual electrolytes. The concentrations of the CNXLs suspensions that were used for multilayer build-up were 5.3 and 5.5 wt % for d-CNXLs and h-CNXLs respectively. Surface Charge Density. The surface charge density of the CNXLs can be calculated from the number of negative charges on the CNXLs surface, related to the number of sulfate groups in the system, and from the nanocrystals dimensions. The charge density of the produced CNXLs was evaluated by both elemental analysis of sulfur content and conductometric titration. The cellulose suspensions were titrated with 0.01 M NaOH using a CDM 210 conductimeter equipped with a CDM 614T electrode. The titration curves showed the presence of strong acid, corresponding to the ionized sulfate groups. The sulfur content of the CNXLs samples determined by conductometric titration and elementary analysis is 0.61 wt %. Considering the nanocrystal dimensions, Elazzouzi et al. recently proposed a detailed investigation of the shape and size of cellulose nanocrystals prepared by acid hydrolysis using TEM, Cryo-TEM, SAXS, and WAXS as complementary techniques.37 These authors have shown that their particles were flat objects constituted of few laterally bound elementary crystallites and not monodisperse cylinders. In the case of cotton as native cellulose source and with a temperature of hydrolysis of 63 °C and a time of hydrolysis of 30 min, the dimensions of the nanocrystals were found to be 128 × 26 × 6 nm3. Taken together, these data finally lead to a charge density of 0.58 e/nm2 for the CNXLs used in this study. LbL Assembly. Multilayers were deposited on polished silicon wafers for neutron reflectivity and AFM experiments. Prior to deposition, the solid substrates were cleaned in a Piranha H2SO4/H2O2 mixture (70:30 v/v, highly corrosive!) at room temperature for 30 min, followed by intensive rinsing with water resulting in a negatively charged surface. Thin films were then prepared using the versatile LbL technique consisting in sequential steps: (i) dipping (35) Gopinadhan, M.; Ivanova, O.; Ahrens, H.; Guenther, J.-U.; Steitz, R.; Helm, C. A. J. Phys. Chem. B 2007, 111, 8426-8434. (36) Nishiyama, Y.; Isogai, A.; Okano, T.; Mueller, M.; Chanzy, H. Macromolecules 1999, 32, 2078-2081. (37) Elazzouzi-Hafraoui, S.; Nishiyama, Y.; Putaux, J.-L.; Heux, L.; Dubreuil, F.; Rochas, C. Biomacromolecules 2008, 9, 57-65.

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of the substrate for 20 min in a polycation aqueous solution (PEI 2 g/L or PAH 4 g/L, NaCl 1M), (ii) rinsing with distilled water for 5 min, (iii) dipping for 20 min in an aqueous PSS solution (4 g/L, NaCl 1M) or CNXLs suspension, (iv) rinsing with distilled water for 5 min. The dipping cycle was then repeated until the desired series of layers has been deposited. After the last rinsing step, the samples were dried for 30 min at 60 °C. The dipping time was chosen equal to 20 min since according to litterature this time should be appropriate to reach adsorption equilibrium. In fact, for the preparation of PDDA/CNXLs multilayers, Podsiadlo et al. have used 10 min adsorption intervals since they have found this to be adequate in many other systems they have prepared previously.29 In a subsequent publication, the same authors built PEI/CNXLs multilayers using the LbL assembly.30 They show that for dipping times as short as a fraction of a second densely covered surfaces were prepared. It has also to be noted that these authors have obtained similar results with other polycations, including PDDA, polyallylamine (PAH), or chitosan. Prior to cellulose nanocrystals deposition, a pure polyelectrolyte multilayer primer, PEI/PSS/(PAH/PSS)n/PAH with n ) 0 or 4, was adsorbed on the silicon substrate. Subsequent multilayer build-up consisted of alternated deposition on the primer of CNXLs and PAH to form PEI/PSS/(PAH/PSS)n/(PAH/CNXLs)m samples with m varying between 1 and 5. Neutron Reflectivity Measurements. Neutron reflectivity is a powerful nondestructive technique giving information on the thickness and composition in the direction perpendicular to the reflecting surface. A major advantage of this technique arises from the possibility to tune the index of refraction using isotopic substitution. Since neutrons are scattered in a significantly different way by hydrogen and its isotope, deuterium, replacing H atoms by D atoms leads to a change in the scattering length density, F, related to the index of refraction, n, by n)1-

λ2 F 2π

(1)

To take advantage of this unique method, the intracrystalline deuteration of cellulose crystals was used.36 Thus, cellulose nanocrystals with an inner partially deuterated core (Fd-cell ) 3.8 10-6 Å-2), denominated d-CNXLs, as well as native protonated nanocrystals (Fh-cell ) 1.9 10-6 Å-2), denominated h-CNXLs, were used in this study. Specular neutron reflectivity experiments were carried out on the time-of-flight reflectometer EROS at the Laboratoire Le´on Brillouin, CEA Saclay, France.38 To access a broad momentum transfer range, data were collected at two different fixed angles, 0.93° and 2°, with a neutron white beam covering wavelengths from 3 to 25 Å. The final accessible Q range is 0.008-0.1 Å-1. Data were analyzed using a , box . model consisting in dividing the thin film into a series of layers. Each layer is characterized by a finite thickness, scattering length density, and interfacial roughness with the neighboring layer. Reflectivity curves calculated using the optical matrix method take into account the angular resolution of the spectrometer. Atomic Force Microscopy. AFM experiments were performed on a Pico plus (Molecular Imaging) commercial instrument. Topography pictures were obtained using tapping mode with Mikromash NSC 36 tips. Data treatment (height measurements after baseline correction only) and presentation were realized with the help of Gwyddion Software.

Results and Discussion Film Characterization after the First Deposition of Cellulose Nanocrystals. Prior to cellulose nanocrystal deposition, a pure polyelectrolyte multilayer primer, PEI/PSS/(PAH/PSS)n/ PAH, was adsorbed on the substrate. It has been previously described that a first layer of PEI is well suited to obtain a uniform (38) Ott, F.; Cousin, F.; Menelle, A. J. Alloys Compd. 2004, 382, 29-38.

Figure 1. Neutron reflectivity curves in RQ4 representation of multilayer samples: pure polyelectrolyte multilayer PEI/PSS/(PAH/ PSS)4/(PAH) (curve a, open circles) and PEI/PSS/(PAH/PSS)4/(PAH/ d-CNXLs)1 (curve c, filled squares). The dashed line (curve b) is the Fresnel reflectivity and the solid lines are best fits as discussed in the text.

coating and favors subsequent multilayer buildup.39 Furthermore, the primer coating reduces the influence of the substrate on CNXLs adsorption. For neutron reflectivity measurements, n was chosen equal to 4 in order to give a thickness that is large enough to fit in the Q range probed. Consequently, there is an increase in the number of observable oscillations (known as Kiessig fringes) related to the thin film thickness, and thus, the data analysis is facilitated and its quality improved. Explicitly, Kiessig fringes are connected to the total thickness, lt, of the multilayer film that can be estimated by lt ) 2π/∆Q, where ∆Q is the spacing between two adjacent minima of the oscillations. The neutron reflectivity curve corresponding to PEI/PSS/(PAH/ PSS)4/PAH is shown in Figure 1 in a RQ4 representation (curve a), which allows us to get rid of the Q-4 decay of the interface and therefore highlights fringes related to the adsorbed layer only. Very well defined Kiessig fringes with maxima close to the Fresnel reflectivity curve (curve b) are observed, reflecting a well-defined layer with a strikingly low roughness. Curve a was fitted using a one-layer model, giving a total thickness of the polyelectrolyte multilayer primer equal to 20 nm in complete agreement with previously described measurements done under the same conditions of ionic strength.40,41 The multilayer film has then been characterized after a first adsorption on the polyelectrolyte primer of cellulose nanocrystals from a suspension resulting from acid hydrolysis of cotton linters. Composition and thickness of the layers were probed by neutron reflectivity, whereas the surface morphology was monitored by AFM. As shown in Figure 2A, a highly uniform surface coverage and very high adsorption density were observed. The isotropic pattern obtained from a Fourier transform of the image indicates that the nanorods have a random orientation (inset in Figure 2A). No influence on surface morphology can be detected when samples with a PAH capping layer and samples with a CNXLs outer layer are compared (data not shown). Very little defects have been observed when imaging large areas (8 × 8 µm2), showing that the LbL technique is well suited for preparing dense and homogeneous cellulose nanocrystals surface if the concentration of the suspension used is greater than or equal to 2 wt (39) Kolasinska, M.; Warszynski, P. Appl. Surf. Sci. 2005, 252, 759-765. (40) Losche, M.; Schmitt, J.; Decher, G.; Bouwman, W. G.; Kjaer, K. Macromolecules 1998, 31, 8893-8906. (41) Von Klitzing, R.; Wong, J. E.; Jaeger, W.; Steitz, R. Curr. Opin. Colloid Interface Sci. 2004, 9, 158-162.

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Figure 3. Neutron reflectivity curves of multilayer samples PEI/ PSS/(PAH/PSS)4/(PAH/h-CNXLs)m with m ) 1, 2, 3, and 4 and of the polyelectrolyte multilayer primer PEI/PSS/(PAH/PSS)4/PAH (noted PE). Solid lines are best fits obtained with the model described in the text.

Figure 2. AFM topography images (4 × 4 µm2) and corresponding 2D fast Fourier transformations of sample PEI/PSS/(PAH/h-CNXLs)1 prepared from the isotropic (A) and the anisotropic (B) phases of a 6.5 wt % CNXLs suspension.

% (Figure S.1 in the Supporting Information). Surface coverage appears significantly more dense and uniform than previously reported CNXLs surfaces obtained by LbL or spin coating.29,31 This high surface density is very different from what was observed with CNTs. AFM studies of LbL films that were first treated with polycations and then with single-walled (SWNT) or multiwalled CNTs (MWNT) reveal uniform adsorption but low surface densities compared to the situation with CNXLs.20,42 As SWNTs possess low surface charge densities, they tend to flocculate, with the result of formation of bundles. In the case of films of CNTs prepared by LbL assembly, the low surface charge leads to slow adsorption and low surface densities. Conversely, for CNXLs, high surface charge of the nanorods and high concentration of the suspension favor fast adsorption and high surface coverage. The neutron reflectivity curve of the sample PEI/PSS/(PAH/ PSS)4/(PAH/d-CNXLs)1 is presented in Figure 1 (curve c). It exhibits very well defined oscillations that are shifted toward lower Q values when compared to the pure polyelectrolyte (42) Olek, M.; Ostrander, J.; Jurga, S.; Mohwald, H.; Kotov, N.; Kempa, K.; Giersig, M. Nano Lett. 2004, 4, 1889-1895.

multilayer PEI/PSS/(PAH/PSS)4/PAH (curve a), showing a significant thickness increase. The amplitude of the oscillations decreases when Q increases, which can be attributed to a roughness increase after nanocrystals adsorption. It has, however, to be noted that such features can only arise from a well defined total thickness of the layer. We observe that the intensity of the first maximum is higher than the Fresnel reflectivity. This effect is due to the use of partially deuterated cellulose nanocrystals, showing scattering length density (SLD) of their crystalline core larger than that of the silicon substrate. Attempts to fit curve c by modeling the CNXLs adsorption by a unique cellulose layer on top of the primer layer were unsuccessful. It was then assumed that the cellulose deposit could be decomposed into two sublayers with the same thickness but different SLDs. A recurring issue with X-ray or neutron reflectivity is the non-uniqueness of the SLD profiles that can be extracted from experimental data since the necessary phase information is incomplete.43 To ensure physical consistency of the parameters extracted from the fitting procedure, partially deuterated and protonated multilayers were modeled independently and both results were compared. The dual layer model for cellulose adsorption gives a good agreement between the calculated and the experimental curves for both PEI/PSS/(PAH/PSS)4/(PAH/d-CNXLs)1 (Figure 1, curve c) and PEI/PSS/(PAH/PSS)4/(PAH/h-CNXLs)1 (Figure 3, m ) 1) with comparable sets of parameters, which is a strong indication of the good reliability of the method. Fitting parameters for both systems are listed in Tables S.I and S.II in the Supporting Information. The fitting procedure indicates that the total cellulose layer is 15 nm thick, i.e., twice that of cotton cellulose nanocrystal thickness, in agreement with ellipsometry measurement performed by Gray et al. for PAH/ cotton CNXLs multilayers.31 Neutron reflectivity results further demonstrate that the two cellulose layers have different adsorption densities: the bottom one is dense since cellulose occupies 50% of the volume and is covered by a more diluted top one where cellulose only occupies 25% of the volume. To complement these results, the height profile of a PEI/PSS/(PAH/h-CNXLs)1 sample, which was recorded by AFM along a 1.2 µm line, is shown in Figure 4. This profile can clearly be divided into two regions. A first 7.5 nm thick region (between h ) 0 and 7.5 nm) on top of the substrate appears densely covered and a second 7.5 nm thick region of reduced density (between h ) 7.5 and 15 nm) is observed on top of the (43) Pershan, P. S. Phys. ReV. E 1994, 50, 2369-2372.

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Figure 4. Height profile of a PEI/PSS/(PAH/h-CNXLs)1 sample recorded by AFM.

first one. Even if a single height scan does not provide strong evidence since it is not statistically representative, the observed features are in full agreement with the adsorption of CNXLs in two layers with different surface densities, as revealed by neutron reflectivity. Oriented Layer. A very interesting property of nanocrystals obtained from sulfuric acid hydrolysis of native cellulose is their spectacular self-assembling into ordered chiral nematic phases above a critical concentration that depends on axial ratio and ionic strength.4,5,44-47 This feature is similar to that of other stable suspensions of biological rodlike colloids, such as tobacco mosaic viruses,48 DNA fragments,49 chitin whiskers50 and fd virus.51 The possibility to use bulk ordering to prepare oriented CNXLs layers was tested by dipping a PEI/PSS/PAH sample into the anisotropic lower phase of a 6.5 wt % biphasic suspension. An AFM image of the surface of the resulting film is shown in Figure 2B. A pronounced degree of orientation is observed and further evidenced by the Fourier transform of the image shown in the inset in Figure 2B. Dipping (20 min) was sufficient to achieve alignment of the cellulose rods. For the biphasic suspension used, the concentration in the anisotropic phase, Ca, must be slightly greater than the total concentration Ct ) 6.5 wt %, i.e., Ca ≈ 7 wt %.4 At this concentration the nanocrystal mobility is high enough to permit a rearrangement leading to the alignment of the chiral nematic director perpendicular to the surface. Cranston and Gray also reported substantial orientation of cellulose nanocrystals deposited using the LbL method from a 9 wt % suspension, but only after a 24 h exposure in a 7 T magnetic field.32 This extended time is probably due to the high concentration used. Multilayered Films. The preparation method was repeated to increase the number of CNXLs layers up to five. To gain insight into the composition of the thin films, neutron reflectivity measurements were thus carried out with samples PEI/PSS/(PAH/ (44) Revol, J. F.; Giasson, J.; Guo, J. X.; Hanley, S. J.; Harkness, B.; Marchessault, R. H.; Gray, D. G. In Cellulosics: Chemical, Biochemical and Material Aspects; Kennedy, J. F., Phillips, G. O., Williams, P. A., Eds.; Ellis Horwood Series Polymer Science and Technology, 1993; pp 115-122. (45) Dong, X. M.; Revol, J. F.; Gray, D. G. Cellulose 1998, 5, 19-32. (46) Araki, J.; Kuga, S. Langmuir 2001, 17, 4493-4496. (47) Orts, W. J.; Godbout, L.; Marchessault, R. H.; Revol, J. F. Macromolecules 1998, 31, 5717-5725. (48) Oster, G. J. Gen. Physiol. 1950, 33, 445-463. (49) Livolant, F.; Leforestier, A. Prog. Polym. Sci. 1996, 21, 1115-1164. (50) Revol, J. F.; Marchessault, R. H. Int. J. Biol. Macromol. 1993, 15, 329335. (51) Dogic, Z.; Fraden, S. Phys. ReV. Lett. 1997, 78, 2417-2420.

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Figure 5. Neutron reflectivity curves of multilayer samples PEI/ PSS/(PAH/PSS)4/(PAH/d-CNXLs)m with m ) 1, 2, 3, and 4 and of the polyelectrolyte multilayer primer PEI/PSS/(PAH/PSS)4/PAH (noted PE). Solid lines are best fits obtained with the model described in the text.

PSS)4/(PAH/h-CNXLs)m and PEI/PSS/(PAH/PSS)4/(PAH/ d-CNXLs)m with m varying between 1 and 4 (Figures 3 and 5). All reflectivity curves show common features and can be divided into two regions. For Q < 0.034 Å-1, Kiessig fringes with positions depending on m are observed. For Q > 0.034 Å-1, one observes Bragg peaks with quasi m-independent amplitude and position. Such features are typical signature of well-ordered films with repeat units assembled to give a well-defined total thickness. Qualitatively, the position of the first minimum of the fringes shifts toward lower Q values when m increases, thus showing an increase of the total layer thickness with the number of deposition steps (Figures 3 and 5). Quantitatively, the evaluation of the position of the following minima appears difficult in some cases since the observed Bragg peak is superposed on the Kiessig fringes. This superposition, when added to the contrast effects, shifts or masks the oscillations of the Kiessig fringes. For this reason, the total thickness of the film was not evaluated from the spacing between the first two minima but the reflectivity data were fitted using a multiple layers model. Both partially deuterated and protonated systems were analyzed separately to ensure physical consistency. Solid lines in Figures 3 and 5 are the best fits obtained respectively in the case of the protonated and the partially deuterated system, showing good agreement with experimental data. Fitting parameters for both systems are enclosed in the Supporting Information. The first basic information obtained from the modeling of the reflectivity is the total thickness of the thin film. From the extracted value, one can calculate the normalized thickness that we define as the total thickness from which one subtracts the primary layer thickness. For comparison, Figure 6 shows the evolution with m of the normalized thickness obtained from both reflectivity data and AFM scratch-height analysis. These data show a linear growth of the film that reflects the regular stacking of the layers. UV/vis spectrometry data (to be reported in a subsequent publication) suggest that this linear thickness increase extends at least up to m ) 10. This behavior is expected for LbL assemblies where little interdiffusion between the oppositely charged species occurs.40,52 It has to be emphasized that such a linear buildup is achieved despite the use of polydisperse building blocks, suggesting compensation of the defects. The thickness increase per deposited (PAH/CNXLs) bilayer is about 16 nm for protonated (52) 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, 1253112535.

Structural Details of Multilayers

Figure 6. Normalized thickness of multilayered films measured by neutron reflectivity for samples PEI/PSS/(PAH/PSS)4/(PAH/dCNXLs)m (open circles) and PEI/PSS/(PAH/PSS)4/(PAH/h-CNXLs)m (closed squares) and by AFM for PEI/PSS/(PAH/h-CNXLs)m (diamonds). The solid line has a slope equal to 16.

cellulose (from reflectivity data or AFM analysis) and of similar value for deuterated cellulose (from reflectivity data). This result indicates that every deposition step results in the adsorption of a CNXLs dual layer divided into the two aforementioned sublayers, as seen after the first cycle. AFM imaging and scratchheight analysis have been performed to characterize the thickness of CNXLs layers after 5, 10, and 20 min dipping time. In all cases, the CNXLs layer was ∼16 nm thick, which suggests an adsorption as a dual layer that is not dipping time dependent in the time range probed. Kontturi et al.33 have reported that single deposition by spin coating from 2 wt % CNXLs suspension results in thick CNXLs films, from 34 to 58 nm, depending on the spinning speed. This observation shows that spin coating and dipping involve different deposition mechanisms, resulting in different properties of the films. Bragg peaks are typically encountered in neutron reflectivity experiments when a superstructure is formed by the repetition at regular intervals of layers with a large contrast difference compared to the neighboring layers.35,40,53 As shown in Figures 3 and 5, the Bragg peak at Q ) 0.0472 Å-1 is much more intense in the case of the deuterated system due to a reinforced SLD contrast between the inner core of each nanocrystal and the surrounding polymer. The position of the Bragg peak should correspond in real space to the thickness lr of the repeat unit of the multilayer. A value of lr about 13.5 nm can be estimated, which is close to twice the lateral size of cotton cellulose nanocrystals. This result shows that the repeat unit of the multilayer consists of a double cellulose layer plus an ∼2 nm thick PAH layer. Reflectivity data analysis thus provides two independent evidence (from fitting of the curves and from position of the Bragg peak) for the presence of dual CNXLs layers repeated at regular intervals to form the multilayer film. The intensity of the Bragg peaks should increase with the square of the number of repeat units and thus be proportional to m2. In our case, however, such behavior is not observed since the intensity of the Bragg peaks does not depend on m. This can be explained by a contrast smoothening effect due to PAH diffusion within the film. As previously mentioned, despite a high surface coverage, the porosity in the CNXLs layer remains large and offers the possibility for PAH macromolecules to diffuse and fill in the gaps between nanocrystals during the dipping (53) Haas, H.; Steitz, R.; Fasano, A.; Liuzzi, G. M.; Polverini, E.; Cavatorta, P.; Riccio, P. Langmuir 2007, 23, 8491-8496.

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Figure 7. Scattering length density profiles extracted from reflectivity data analysis for samples PEI/PSS/(PAH/PSS)4/(PAH/ d-CNXLs)m with m ) 1, 2, 3, and 4 and of the polyelectrolyte multilayer primer PEI/PSS/(PAH/PSS)4/PAH (noted PE). In the inset, profiles for PEI/PSS/(PAH/PSS)4/(PAH/d-CNXLs)m (open circles) and PEI/PSS/(PAH/PSS)4/(PAH/h-CNXLs)m (closed squares) are compared for m ) 1 and 2.

procedure. This diffusion of polycation chains leads to a change in composition of the cellulose layer that is now constituted of CNXLS, PAH, and air, resulting in a decreased value of the SLD of the layer. This reduction of the contrast between deuterated cellulose layers and PAH neighboring layers together with a larger roughness prevent any increase in Bragg peak intensities when m increases. From the neutron reflectivity analysis, one can plot the SLD profiles, related to the evolution of the composition along the normal to the multilayer film surface (Figure 7 and inset). As shown in the inset, comparable geometrical parameters (thickness and roughness) were found for two different isotopic compositions of the same physical system (deuterated and protonated multilayers for m ) 1 and 2), showing the validity of the model. The profiles shown in Figure 7 can be divided into different regions. First, PEI/PSS/(PAH/PSS)4/(PAH/d-CNXLs)m multilayers exhibit a plateau in the 0-200 Å z range corresponding to the polyelectrolyte primer layer. In this region, SLD variations are due to different hydration degrees of the polyelectrolytes but do not affect the estimated thickness. In the 200 Å < z < 350 Å region, the curves show a peak corresponding to the first CNXLs double layer. The peak maximum is 1.9 10-6 Å-2 for m ) 1 and decreases with m. This maximum is then followed by a slow decrease of the SLD over a z range that increases with m. The final region shows a rapid decay standing for the multilayer-air interface characterized by the external roughness, σext. Contrary to pure polyelectrolyte (PSS/PAH) systems with equally spaced deuterated PSS layers, no oscillation in the SLD profiles is observed despite the use of deuterated cellulose nanocrystals. Together with the decrease with m of the maximum in the 200350 Å region, this absence of oscillations can be attributed to an increasing degree of interdigitation of the layers with m that results in a flattening of the SLD. The slope of the final decay at the film-air interface decreases with m due to increasing external roughness. Similarly, the AFM image of sample PEI/ PSS/(PAH/d-CNXLs)5 (Figure S.2 in the Supporting Information) exhibits some thickness fluctuations and shows more defects than sample PEI/PSS/(PAH/d-CNXLs)1 presented in Figure 2A. The external rms surface roughness measured by AFM is about 4 ( 1.5 nm for m ) 1 and 7.5 ( 1.5 nm for m ) 5. These observations indicate that not only the degree of coarsening increases with the number of adsorbed CNXLs layers but also

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that relatively smooth surfaces are preserved since roughness values below 10 nm are measured. Mechanisms of Formation of the CNXLs/PAH Multilayer. Polyelectrolyte multilayer formation is governed not only by an enthalpy decrease due to complex formation when electrostatic attraction between species of opposite charge occurs, but also by entropy increase due to counterions release together with the liberation of solvent molecules from the solvation shell of the polymer-bound ionic groups.41,54,55 As cellulose nanocrystals are negatively charged rods (a part of the surface hydroxyl groups have been replaced by sulfate groups during acid hydrolysis with H2SO4), this gain of entropy is associated with the counterions release. If the adsorption of the first layer of the negatively charged CNXLs on the positively charged PAH surface is favorable on enthalpy consideration, this is not the case for the second layer that directly adsorbs on another negatively charged CNXLs layer. The adsorption is thus necessarily entropy driven. The entropy gain associated with counterions is indeed of a strong importance as it overcompensates the loss of conformational entropy of polymers chains due to adsorption in classical polyelectrolyte multilayer formation. This entropy gain is a priori maximum when all positive charges from the PAH surface are compensated by negative charges from CNXLs, i.e., if the charge reversal of the surface is reached in the system. This hypothesis can be tested by comparison of the charge densities of both components of the system. On the basis of polyelectrolyte mass densities measured by neutron reflectivity,40 the PAH charge density was estimated by Cranston and Gray to be 3.00-3.75 e/nm2 depending on the hydration of the film.31 As described in the Experimental Section, the surface charge density of the CNXLs can be estimated to 0.58 e/nm2. The comparison of the two values immediately shows that, even for a full coverage of the PAH surface by one layer of CNXLs nanocrystals, charge reversal could not be reached. This difference favors the adsorption of a double cellulose layer to maximize the entropy gain. Charge reversal is usually a prerequisite for multilayer buildup in polyelectrolyte systems each time a new layer is deposited. In our case, however, adsorption of PAH after the adsorption of two layers of CNXLs and subsequent multilayer growth occurs even when charge reversal is not reached. Here, this may be possible because the outer surface of the CNXL nanocrystals most probably remains negatively charged after adsorption on the PAH surface. A compensation of the charge of the outer surface of the CNXLs would indeed mean either the formation of large loops of PAH or the desorption of polycation chains from the inner surface. These two situations are highly improbable since the distance between the cellulose outer surface and the PAH layer is high (15 nm) and because of the strong interaction between the PAH layer and the underlying PSS or CNXLs layer. This original situation can be described as a surface charge reversal, whereas the bulk charge reversal cannot be complete. As already mentioned, PAH molecules can diffuse within the film and weaken the contrast between adjacent layers. Still, despite this blurring effect connected with polycation penetration, a welldefined total thickness of the film is preserved and a linear growth of the multilayer is observed. It can tentatively be assumed that (54) Bertrand, P.; Jonas, A.; Laschewsky, A.; Legras, R. Macromol. Rapid Commun. 2000, 21, 319-348. (55) Schonhoff, M. J. Phys.: Condens. Matter 2003, 15, R1781-R1808.

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flexible polycation chains tend to wrap around highly rigid cellulose nanocrystals to achieve charge compensation and counterion release. PAH molecules also help covering irregularities favoring the linear buildup of the multilayer. It is also worth mentioning that in the case of LbL assembly of nanoparticles, strong Van der Waals attraction forces between components can participate in uniform growth of films. As shown by AFM measurements and SLD profiles, a moderate roughness increase is observed when the number of CNXL layers is increased. In the m ) 1 situation, the first CNXLs, which are deposited to form the lower sublayer of the double layer, are likely to lay flat since these negatively charged nanorods are adsorbed on a positively charged polyelectrolyte base, presenting a very smooth surface (rms surface roughness smaller than 1 nm). When this initial surface is covered by CNXLs, the resulting new surface displays a somewhat increased roughness. This is not only due to the inevitable voids resulting from the imperfect lateral packing of the nanorods but also to the thickness polydispersity of the rods themselves. Thus, when further nanorods are deposited to create the upper sublayer of the first CNXL layer, they no longer lay flat but adopt a somewhat jumbled organization leading to an increased external film roughness. A consequence of this disorganization is that the PAH interlayer, required for the next CNXLs layer build up, will no longer be a flat surface, even if the deposition of this polyelectrolyte tends to diminish the irregularities of the underlying CNXLs layer. Starting therefore from an already rough surface, the subsequent layers of CNXLs will become more disorganized than the first one and this phenomenon will increase at every layer, when m is increased.

Conclusion The combination of AFM measurements and neutron reflectivity experiments have allowed us to give a detailed characterization of thin films composed of alternating layers of rigid cellulose nanorods and flexible polycation chains. Both components interact to give well-ordered films exhibiting a linear thickness increase with the number of layers and reduced surface roughness. We believe that the film buildup is mostly governed by entropy with PAH flexibility playing a key role to preserve linear growth and surface smoothening. The possible alignment of the nanocrystals opens the route to a finer control of the architecture of multilayers. It will potentially lead to the buildup of bio-inspired films mimicking the organization of cellulose microfibrils in living organisms such as in wood and could thus be applied to the rational design of new high performance materials. Acknowledgment. We thank the Innovation Centre for Nanobiotechnologies (Nanobio, Grenoble) for granting access to the AFM facilities. We are also grateful to H. Chanzy (CERMAV) for valuable help during the editing of the manuscript. Supporting Information Available: Tables listing the parameters obtained from the fitting procedure of the reflectivity curves for samples PEI/PSS/(PAH/PSS)4/(PAH/d-CNXLs)m and PEI/PSS/(PAH/ PSS)4/(PAH/h-CNXLs)m for m ) 1-4, as well as for the polyelectrolyte multilayer primer. These parameters were used to plot the SLD profiles shown in Figure 7. It also contains 8 × 8 µm2 AFM images of samples PEI/PSS/(PAH/h-CNXLs)1 and PEI/PSS/(PAH/d-CNXLs)5. This material is available free of charge via the Internet at http://pubs.acs.org. LA703045F