Article pubs.acs.org/Biomac

Chitin Nanocrystal-Xyloglucan Multilayer Thin Films Ana Villares,* Céline Moreau, Isabelle Capron, and Bernard Cathala UR1268 Biopolymères Interactions Assemblages, INRA, F-44316 Nantes, France S Supporting Information *

ABSTRACT: For the first time, the adsorption of xyloglucan (XG) on chitin nanocrystals (ChiNC) surface was proved using quartz crystal microbalance with dissipation (QCM-D) and by successfully building up spin-coated assisted layerby-layer (LbL) structures on solid substrates. Several parameters in the adsorption process, such as ChiNC concentrations (0.5−3.0 g L−1), number of layers, or the outmost layer material (ChiNC or XG), were investigated to better understand the fabrication process of multilayer films. The thickness of the homogeneous film increased linearly with the number of bilayers, with an average thickness per bilayer of 12.3 nm. Additionally surface morphology was studied by atomic force microscopy (AFM), which revealed an almost completely covered surface after the adsorption of ChiNC. The final structures were found to have semireflective properties capable of being tuned by adjusting the ChiNC dispersion parameters.



that have β-(1→4)-linked anhydroglucose cellulose-like backbone decorated with α-D-xylosyl and β-D-galactosyl moieties. In vivo XG-cellulose interaction is thought to be a key element of the load bearing network of plant cell wall. The exact nature of the binding of XG to cellulose remains matter of debate in literature but it was described to occur through concomitant contributions of van der Waals forces, polar interactions, and hydrogen bonding.15,16 Furthermore, molecular modeling investigations have pointed out that interactions between XG and cellulose are not specific certainly due to the involvement of several attractive forces.15,16 This lack of specificity may thus offer opportunities to XG to interact with other surfaces and possibly with ChiNC nanocrystals. Chitin and chitin derivatives were also the matter of several researches aiming to explore the fabrication of chitin-based assemblies by the alternate deposition of chitin and a wide variety of polysaccharides. The composites containing chitin or chitosan, obtained by deacetylation of chitin, have been widely studied due to their potential biological properties, including antimicrobial activity,17,18 and the possibility to act as active packaging supports for several applications such as wound dressing materials,19 or drug delivery systems,20 among others. Multilayer films have been prepared with chitosan and alginate,21 chitosan and heparin,22−24 or even modified chitosan and hyaluronic acid.25 Similarly, the fabrication of chitosan/ cellulose molecularly engineered nanocomposites has been intensively studied.26−28 Very recently, negatively charged CNC and positively charged chitin nanocrystals (ChiNC) have been used to elaborate multifunctional coatings obtained by the layer-by-layer (LbL) method.29 All the multilayer growth cited above are driven by electrostatic interactions since chitin/ chitosan acts as a weak polycation; nevertheless, secondary

INTRODUCTION The fabrication of nanostructured coatings has attracted much attention in recent years due to the possibility of modifying or improving the characteristics of surfaces by the deposition of thin films. The use of natural polymers including polysaccharides, polypeptides, and proteins has been a matter of research because of their abundance, low toxicity, biodegradability, and biocompatibility. In this field, polysaccharides comprise a wide range of structures capable of being assembled into highly ordered architectures. In nature, cellulose and chitin are the two most abundant polysaccharides that occur as fibrilar structures providing mechanical properties to living organisms. These two linear polysaccharides present some structural analogy since their backbones are similar and they occur as a crystalline structure presenting ordered fibrous organization. Cellulose is composed of β-(1→4) anhydroglucose, while chitin is a polymer of β-(1→4) linked N-acetyl anhydroglucosamine units. Fibrils of chitin and cellulose are constituted by crystalline and amorphous zones that can be hydrolyzed to isolate stiff crystalline nanorods referred as whiskers or nanocrystals.1,2 Hydrolysis provides negatively charged sulfate groups to the cellulose nanocrystals surface, which therefore act as anionic polyelectrolytes. Differently, the amino groups confer positively charged surface to chitin nanocrystals so that they behave as weak polycations. Cationic polysaccharides are scarcely found in nature; therefore, chitin nanocrystals might provide a different building moiety for the fabrication of new architectures. Both chitin nanocrystals (ChiNC) and cellulose nanocrystals (CNC) have been intensely used for the elaboration of materials3 as well as for the construction of multilayer thin films based on electrostatic interactions of CNC in alternation with synthetic polyelectrolytes.4−8 Efforts have also been made to elaborate multilayer structures fully composed of natural polymers. This has been notably achieved by taking advantage of the striking affinity of xyloglucans (XG) to the cellulosic surface.9−14 XG are neutral polysaccharides © 2013 American Chemical Society

Received: October 2, 2013 Revised: December 4, 2013 Published: December 11, 2013 188

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procedure but the time for adsorption of water was 10 s. One bilayer is defined as a single deposition step of ChiNC and an adsorption step with XG, each followed by a rinsing step. A bilayer is represented as whole numbers (m), differently from the fractionated numbers, which indicate that ChiNC is the outermost layer. Quartz Crystal Microbalance with Dissipation (QCM-D). The QCM-D measurements were performed with a Q-Sense E4 instrument (AB, Sweden) using a piezoelectric AT-cut quartz crystal coated with gold electrodes on each side (QSX301, Q-Sense). All measurements were carried out at 20 °C using the QCM flow cell modules. The goldcoated crystals were excited through a driving voltage applied across the gold electrodes at its fundamental frequency ( f 0 = 5 MHz) as well as at the 3rd, 5th, 7th, 9th, 11th, and 13th overtones (n = 3, 5, 7, 9, 11, and 13, respectively) corresponding to 15, 25, 35, 45, 55, and 65 MHz, respectively. Any material adsorbed on the crystal surface induces a decrease of the resonance frequency (Δf). If the adsorbed mass is evenly distributed, rigidly attached and small compared to the mass of the crystal, Δf is directly proportional to the adsorbed mass (Δm) using the Sauerbrey’s equation:34

interactions including hydrogen bonds and hydrophobic interactions may also play an important role in the formation and structure of the films.29 To follow up the efforts to obtain entirely biobased materials, we have investigated the interaction of ChiNC and XG to fabricate nanostructured thin films taking advantage of the structural analogy existing between CNC and ChiNC. This article describes the fabrication of multilayer films containing chitin nanocrystals and xyloglucan investigated by QCM-D and by spin-coated assisted LbL procedure. The growth pattern was monitored by means of ellipsometry to obtain values of thickness and by measuring contact angle to get more insight into the wetting properties. Additionally surface morphology was studied by atomic force microscopy (AFM). Several parameters were investigated to better understand the film build-up including ChiNC concentration, the number of deposited layers, and the outmost layer material. The semireflective properties of the films leading to structural colors are described.



Δm = − C

EXPERIMENTAL SECTION

Δf n

(1)

where C is the constant for the mass sensitivity of the quartz crystal (0.177 mg m−2 Hz−1 at f 0 = 5 MHz) and n is the overtone number. Strictly speaking, the Sauerbrey’s relation is not valid in liquids, especially for adsorbed polysaccharides for which the energy dissipated to the environment is greatly increased due to coupled water. Dissipation signals (ΔD) are recorded simultaneously to frequency shifts and they provide a measure of the frictional losses due to the viscoelastic properties of the adsorbed layer. In this way, the relative stiffness or conformation of the adsorbed layer can be evaluated. High dissipation values reflect a thick and loose adsorbed layer, whereas a thin and rigid layer that vibrates with the crystal is characterized by a low dissipation factor. A baseline was first established by continuously flowing pure water solution on the quartz crystal surface, then frequency and dissipation signals were offset to zero just before injection of polymer solutions in a continuous mode at a flow rate of 0.1 mL min−1. ChiNC dispersion (at 0.5, 1.0, or 3.0 g L−1) and XG solution (at 0.5 g L−1) were alternately injected into the QCM-D cell until a plateau value of frequency and dissipation signals was reached. Then, a rinsing step of the surface with 5 mM NaCl solution or pure water for ChiNC or XG, respectively, was performed before the subsequent injection of polymer solution. QCM-D experiments were carried out twice. The normalization was done by dividing the frequency and dissipation by the corresponding harmonic number (Δf n/n and ΔDn/n). Microscopy Characterization. Transmission Electron Microscopy (TEM). ChiNC suspensions in water were deposited on freshly glow-discharged carbon-coated electron microscope grids (200 mesh, Delta Microscopies, France) and the excess of water was removed by blotting. The sample was then immediately negatively stained with uranyl acetate solution (2%, w/v) for 2 min and dried after blotting at 40 °C just before observation. The grids were observed with a Jeol JEM 1230 TEM at 80 kv. AFM Measurements. Surface observation on silicon wafers was carried out by atomic force microscopy (AFM) by means of an Innova AFM (Bruker). The images were collected in tapping mode under ambient air conditions (temperature and relative humidity) using a monolithic silicon tip (TESPA, Bruker; spring constant k = 42 N/m; frequency f 0 = 320 kHz). Image processing was performed with the WSxM 5.0 software. The roughness was determined from the rootmean-square (RMS) value of 2 × 2 μm2 scan area. Variable-Angle Spectroscopic Ellipsometry. Thickness of multilayer films was evaluated using a variable-angle spectroscopic ellipsometry (M-2000U; J.A. Woollam, Lincoln, U.S.A.). The ellipsometric angles, Δ and Ψ, were acquired over the spectroscopic range 250−1000 nm at three angles of incidence 65, 70, and 75°. Optical modeling and data analysis were performed using the CompleteEASE software package (J.A. Woollam Co., Inc.) using a

Materials. Chitin flakes extracted from crab shells were purchased from France Chitin. Xyloglucan (XG, Mw = 202 kDa) from Tamarindus indica was provided by Megazyme (Ireland). Aqueous xyloglucan solutions were prepared at a concentration of 0.5 g L−1 with deionized water (18.2 MΩ, Millipore Milli-Q purification system) without pH adjustment. Silicon wafers and gold-coated quartz crystals were used for film construction for ellipsometry and QCM-D experiments, respectively. Both solid supports were cleaned in piranha solution H2SO4/H2O2 (7:3, v/v), rinsed thoroughly with Milli-Q water, and dried with a stream of nitrogen. QCM-D quartz sensors were subjected to a plasma etching device (Harrick Plasma) prior to use. Preparation of Chitin Nanocrystals. ChiNC were prepared from chitin flakes extracted from crab shells following a protocol inspired by Revol et al.30 Briefly, 4 g of chitin were submitted to acid hydrolysis in 80 mL of boiling HCl 3 N for 90 min. The resulting suspension was washed with Milli-Q water by successive centrifugations during 20 min at 10000 rpm and dialyzed against HCl 0.01 M for 5 days. The suspension was sonicated (QSonicaSonicator) in order to disperse the remaining aggregates with intermittent cycles for a total operating time of 10 min and successively filtrated through a 5 and 1.2 μm pore sizes cellulose nitrate membranes (Sartorius). The ChiNC charge (0.483 ± 0.020 e nm−2) was determined following the procedure described by Raymond et al.31 by the conductometric titration with freshly prepared 10 mM NaOH solution on a TIM900 titration manager (Gemini B.V., Apeldoorn, Netherland) and a CDM230 conductivity meter (Radiometer Analytical SAS, Villeurbanne, France) equipped with a CDC749 titration cell (Radiometer, Denmark). ChiNC dispersions were adjusted at pH 3 and the stock suspension was then kept at 4 °C. For the film fabrication, ChiNC dispersions at a concentration of 0.5− 3.0 g L−1, as determined by measuring the dry weight, were prepared in 5 mM NaCl and HCl at pH 4.0 so that the glucosamine residues were almost fully protonated.32 In order to weaken intermolecular interactions, chitin nanocrystals were ultrasonicated (QSonicaSonicator) by immersing 1−2 mm of the probe into ChiNC dispersion and applying an amplitude of 1 at 20 kHz for 20 s. Dispersions were filtered (5.0 μm) prior to use so as to remove the remaining traces of aggregates.33 Spin-Coating Procedure. Polymer multilayer films were assembled on polished silicon (100) wafers purchased by WaferNet, Inc. (San Jose, U.S.A.). Spin-coated films were fabricated with a homemade spin coater. The ChiNC dispersion and XG solution were alternatively dropped on the substrate by a four-channel peristaltic pump. The silicon wafer was accelerated at 60 rpm s−1 after 5 min of adsorption and spun at 3600 rpm for 60 s. The films were rinsed with water after every layer was fabricated using the same deposition 189

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obtained with average dimensions of 160 ± 77 nm in length and 16 ± 5 nm in width. Chitin, which is a poly-N-acetyl-Dglucosamine, is never fully acetylated, and some amino groups are present randomly distributed on the surface. We assume that only the NH3+ groups present on the surface are the origin of the electrostatic repulsive force in the suspension.31 The surface charge density was calculated using conductometric titration and found to exhibit a positive surface charge of 0.483 ± 0.020 e nm−2 when fully protonated. This value was in accordance with that given by Li et al.37 Chitin nanocrystals showed high tendency to form aggregates in acidic media independently of the proportion and distribution of acetylated and nonacetylated residues of chitosan. Such aggregates are formed mainly due to intermolecular interactions by hydrogen bonds and by hydrophobic interactions between the acetyl groups. It was therefore necessary to thoroughly optimize the chitin dispersion conditions in order to minimize the formation of molecular aggregates. QCM-D Study of XG Adsorption of ChiNC Surfaces. In situ quartz crystal microbalance with dissipation (QCM-D) was used in order to investigate possible adsorption of XG on ChiNC surface. The ChiNC dispersions and XG solution were sequentially injected onto the quartz crystal surface in a continuous flow until the signals of frequency (Δf) and dissipation (ΔD) remained stable, allowing polymer adsorption to reach equilibrium. A rinsing cycle with 5 mM NaCl solution or pure water was performed after ChiNC and XG adsorption, respectively, in order to remove all polymers that were loosely bound to the surface. Deposition was studied at three ChiNC concentrations (0.5, 1.0, and 3.0 g L−1), whereas XG concentration was kept at 0.5 g L−1 because rheological investigation revealed that this concentration corresponded to a semidilute regime of XG solution where the polymer chains were not entangled and, consequently, the multilayer growth was expected to be linear.12 The evolution of normalized frequency (Δf n/n) and dissipation (ΔDn/n) signals for overtone number n = 3 as a function of time for ChiNC dispersions at 0.5, 1.0, and 3.0 g L−1 and for XG solution at 0.5 g L−1 is shown in Figure 2. The decrease of frequency signal (Δf n/n) after each polymer injection, concomitantly with an increase of dissipation signal, evidenced that hydrated mass (polymer with coupled water) was being adsorbed at the crystal surface. Complete adsorption was assessed when the plateau of frequency and dissipation

three layers model consisting on the Si(100) substrate layer, a thin SiO2 layer, and the single Cauchy layer that describes the multilayer (ChiNC-XG) film. The Cauchy parameters for each sample were used to model the ellipsometry data from the multilayer films to obtain their respective thicknesses. Average thickness value was obtained from measurement of at least six spots per film. Contact Angle Measurement. The static contact angle of water on films was measured by means of a Digidrop Contact Angle Meter (GBX Scientific Instruments) using the sessile drop method. A 5 μL water drop was made on the tip of a syringe and placed on the substrate surface by moving the substrate vertically until contact was made between the drop and the sample. High-resolution images of the droplets were captured via a video camera and Digidrop software was used for data collection and analysis. Six individual measurements were obtained for each set of data and the average values are calculated.



RESULTS AND DISCUSSION Chitin Nanocrystals. Chitin nanocrystals (ChiNC) can be extracted from biological tissues by hydrolysis with HCl 3 N in boiling conditions.30,35,36 This treatment allows to hydrolyze regions of low lateral order so that the water-insoluble, highly crystalline residues are preserved, and when dispersed in aqueous media they form colloidal suspensions. Electron microscopy was used to visualize and measure the dimensions of such ChiNC based on 280 individual measurements. Figure 1 shows a TEM micrograph of the elongated nanoparticles

Figure 1. TEM micrograph of negatively stained chitin nanocrystals obtained by acid hydrolysis of crab shells.

Figure 2. Frequency (Δf n/n) and dissipation (ΔDn/n) changes for the overtone number n = 3 during adsorption of ChiNC dispersion at 0.5 g L−1 (○), 1.0 g L−1 (□), and 3.0 g L−1 (Δ) and XG solution (0.5 g L−1) on the gold surface of the QCM-D sensor as function of time. Arrows indicate the injection of the polymers (ChiNC and XG) and the rinse steps (NaCl or H2O) in between. 190

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The process was only monitored for the deposition of one bilayer of (ChiNC-XG) since after this point highly hydrated films were rather thick to be controlled by the QCM-D38 and the multilayer formation required a drying step to be efficient as we have previously reported for poly(allylamine)-CNC thin films.8 The water removal leads to denser polymer layers and favors the interaction between polymers by nonspecific interactions such as hydrogen bonds. This was the reason why the procedure chosen for the construction of films with a higher number of deposits was inspired from those used in our previous work and included a drying step (i.e., during the spinning) after ChiNC and XG adsorption.8,12 The results presented here clearly demonstrated that XG interacts with ChiNC, reinforcing the idea reported by previous molecular modeling experiments that XG association with polysaccharide surface is not linked to specific moieties. The adsorption process of XG on the cellulose surface has been extensively studied and has been demonstrated both experimentally and by molecular modeling approaches. The later conclude that XG adsorption occurs through a combination of electrostatic, van de Waals interactions and hydrogen bonds.16 The influence of water has also been investigated since it is known to affect the polysaccharide conformations and to be adsorbed on cellulose surfaces. In the presence of water, van der Waals forces were found to be the main driving forces, while the electrostatic contribution was decreased in comparison to studies achieved in vacuum.15 Accordingly, it can be supposed that XG/ChiNC association occurs through similar driving force as XG/cellulose ones, that is, van der Waals forces, polar interactions, and hydrogen bonding. Previous work conducted on adsorption of chitosan and cellulose have also pointed out the similarity between cellulose and chitosan backbone that may promote nonelectrostatic interactions.39 Nevertheless, complete description of XG/ChiNC interactions/association remains to be done and will be the topic of a future report. LbL Deposition. Chitin nanocrystals (ChiNC) and xyloglucan (XG) were alternatively deposited onto solid supports by using the spin-assisted LbL method according to the following procedure: (i) the ChiNC dispersion was dropped onto a silicon surface and allowed to adsorb for 5 min; (ii) then spinning occurred, ejecting the excess of solvent and excess materials, achieving the drying of the surface; (iii) the deposited layer was afterward rinsed with water to remove all loosely bound polymers; and (iv) XG was subsequently dropped onto the substrate, adsorbed for 5 min and spun, followed by identical rising step. In a first approach, the influence of concentration of ChiNC dispersions was investigated by the elaboration of 8-bilayer films. Figure 3 shows the variations in the thicknesses measured by ellipsometry of the (ChiNC-XG)8 films prepared using different ChiNC concentrations (0.5, 1.0, and 3.0 g L−1). As the figure plots, the multilayer thickness showed a clear linear dependence upon ChiNC concentration. Since XG concentration remained constant from one sample to another, the differences in film thicknesses among the three studied concentrations are attributed to ChiNC deposition. The average increment per bilayer can be estimated to be 9.0, 12.3, and 24.0 nm with increasing ChiNC concentration from 0.5 to 3.0 g L−1. This clearly suggests an evolution of the interactions between ChiNC inducing either a change in the surface coverage or in the deposition of multilayer ChiNC structures at higher concentrations. Films consisting of a

signals was reached. Subsequent rinsing did not induce significant frequency or dissipation changes, suggesting that the polymers were irreversibly attached to the surface, as also confirmed by the two injection-rinsing cycles performed for ChiNC and XG at the three ChiNC concentrations studied. The frequency decreases seemed to indicate that the adsorbed mass of ChiNC was in the same range for the three ChiNC concentrations (0.5, 1.0, and 3.0 g L−1) even though the 3.0 g L−1 was slightly higher. In the same way, XG adsorption onto ChiNC surface was revealed and led to similar value of frequency decrease (118.65 ± 4.43 Hz) for the three concentrations of ChiNC indicating that the amount of adsorbed XG onto ChiNC surface was optimum from low ChiNC concentrations (0.5 and 1.0 g L−1). These results suggest that surface “saturation” was achieved from the first polymer injection and that polysaccharides were irreversibly attached to the surface since neither further injections of the like polymer nor rinsing step significantly modified Δf n/n and ΔDn/n. By applying the Sauerbrey’s equation, a higher adsorbed mass of ChiNC (36.1 ± 3.2 mg m−2) than of XG (20.9 ± 0.6 mg m−2) for every bilayer could be estimated. As QCM-D also measures the water bound to the attached polysaccharides, the adsorbed mass estimations could not be representative of the effective adsorbed mass of ChiNC or XG and the presence of high water content on films has likely to be considered. Indeed, the separation of overtones throughout the whole range after polymer adsorption accompanied by the increase of ΔD/n clearly reflected a nonrigid and viscoelastic character of the film, typical of soft and hydrated films. The changes of Δf n/n and ΔDn/n signals for overtone numbers n = 3, 5, 7, and 9 are shown in the Supporting Information (Figures S1−S3). In the particular case of the ChiNC at 3 g L−1 experiment, when the dispersion was injected again, Δf n/n signal decrease suggested adsorption on the surface. However, when rinsing solution was applied, the Δf n/n signal increased to the previous level indicating the removal of loosely bound materials. This fact may indicate that interactions between ChiNC in suspension and the associated surface adsorption processes are influenced by the concentration of ChiNC dispersion. This point will be confirmed later by the evaluation of the thickness of 8 bilayer thin films constructed with different ChiNC concentration (Figure 3).

Figure 3. Influence of chitin nanocrystals (ChiNC) concentration on the thickness measured by ellipsometry of 8-bilayer (ChiNC-XG) films. Photographs of the corresponding films are showed. Error bars are smaller than symbols. 191

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polymer adsorption. At each layer, the plot displayed a staircase-like behavior since the thickness increment varied depending on the polymer deposited. Due to its flexible nature, XG may adopt a more highly swollen random coil conformation, which leads to thinner layers. This behavior has been previously reported for cellulose nanocrystals/ xyloglucan multilayer films.11 AFM. Figure 5 shows the AFM topography images from one layer of ChiNC and 8-bilayer (ChiNC-XG) films at 1.0 g L−1 ChiNC and 0.5 g L−1 XG deposited onto silicon wafers. Representative cross-sectional profiles of the surfaces are included. The AFM topography images and cross-sectional profiles of the ChiNC and (ChiNC-XG)8 films prepared with ChiNC concentrations of 0.5 and 3.0 g L−1 are shown in Supporting Information (Figures S4 and S5). The images from one layer of ChiNC clearly showed the rodlike structure of ChiNC. The size was 180 ± 50 nm in length, in good agreement with the morphology determined by TEM. The topography images revealed that the surface was almost totally covered by the ChiNC layer. The bare silicon domains allowed the determination of film thickness. Hence, the analysis of the cross-sectional profiles showed that the thickness was approximately 15 nm. This value was in good agreement with the fitting results of the ellipsometry data. The roughness of the ChiNC layer was calculated as the root mean squared (RMS) roughness and the obtained value was 6.5 nm. Upon multilayer deposition, the 8-bilayer (ChiNC-XG) films showed similar features as the one-layer ChiNC films, which indicated that the molecular architecture was maintained after the successive layer adsorption. The images revealed high density and uniform surface coverage with low RMS roughness (9.3 nm). Wetting Properties. Figure 6 shows the surface wettability changes measured as the water contact angle of the (ChiNCXG)m films as a function of the number of layers. The contact angle systematically and reproducibly alternated from higher to lower values as the outermost layer was ChiNC or XG, respectively. This oscillation magnitude in the contact angle was gradually smeared out up to the five layers, and then it reached a plateau at about 37°. The rather rigid structure of ChiNC as solid particles may result in the disordered stacking of nanocrystals and therefore the formation of nanoporous surfaces containing air that is hydrophobic. This assumption was confirmed by the AFM images, which clearly showed how the patterns of ChiNC structure could be observed even after the deposition of eight bilayers. Based on this suggestion, the subsequent adsorption of xyloglucan on ChiNC surface may be driven by partial penetration of XG molecules into the pores between ChiNC fibrils. This fact could be explained in terms of the flexible nature of XG, which provides some degree of conformational freedom compared to stiff ChiNC nanorods. Also, the ability of xyloglucan to get into the pores on the ChiNC surface would modify the roughness of the surface and this effect of wettability should be considered. Moreover, the higher contact angles when ChiNC was the outermost layer indicated that ChiNC may also result in a more hydrophobic nature than XG, which has been ascribed to the N-acetyl groups at C2 position29 and has been previously reported for LbL films containing chitin derivatives.44 These results further support the notion that the outermost adsorbed layer distinctly influences the wettability of the surface and that it takes only one layer to achieve the surface modification provided by the bilayer. Chitin nanocrystals have demonstrated to be excellent candidates for the fabrication of multilayer films. The properties

combination of ChiNC and XG showed the development of clear interference colors, as Figure 3 shows. Light reflecting from the air/film surface undergoes constructive and destructive interference with light reflected at the underlying polymer/substrate interface, leading to selective wavelength reflection depending on the film thickness.40 The occurrence of colored films has been previously described for CNC/XG systems13,14,41 and for chitosan and its modified derivatives;42,43 nevertheless, as far as we are concerned, this is the first report to describe this effect in the (ChiNC-XG) system. In this case, the color changed with ChiNC concentration as a consequence of different thickness, turning from light brown to blue and finally to purple and yellow over the concentration range studied. Therefore, semireflective properties of films changed as a result of film thickness. At low ChiNC concentrations (0.5 g L−1), the surface of the 8-bilayer film was light brown (72.6 nm), whereas it turned to blue at 1.0 g L−1 (98.9 nm) and to purple-yellow at 3.0 g L−1 (196.9 nm). These results suggested the possibility of preparing new biofilm composites leading to structural colors tunable by modulation of the concentration of the ChiNC dispersion. In order to get more insight into the build-up process, the thickness after single layer deposition was investigated. The concentration of chitin dispersions was set at 1.0 g L−1 since QCM-D results suggested that surface saturation was achieved and the photograph revealed a homogeneous totally covered surface in this condition. Figure 4 depicts the evolution in

Figure 4. Growth in layer thickness of the (ChiNC-XG)m films at 1.0 g L−1 ChiNC and 0.5 g L−1 XG as a function of the number of bilayers, m, deposited on a silicon wafer, as measured by ellipsometry. Empty squares (□) indicate ChiNC deposition and filled squares (■) correspond to XG deposition. Error bars are not visible since they are too small compared to symbol size.

thickness measured by ellipsometry of the (ChiNC-XG) system for ChiNC dispersions at 1.0 g L−1 and for XG solution at 0.5 g L−1. A bilayer is defined as the film composed of a ChiNC layer and an outermost XG layer and it is represented as whole numbers, differently from the fractionated numbers, which indicate that ChiNC is the outermost layer. Multilayer films showed a linear increase in thickness with the number of deposited bilayers, which clearly indicated a stable and replicable growth process since all the data reported here arise from independent experiments (i.e., each measure corresponds to one sample elaborated independently to the other ones). The QCM-D results have previously discarded the possibility of nonspecific deposition forced by spinning the substrate; therefore, the thickness increase must be ascribed to 192

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Figure 5. Tapping-mode AFM images and the corresponding profiles of the surfaces of one ChiNC layer (a) and 8-bilayer (ChiNC-XG) films (b) at 1.0 g L−1 ChiNC and 0.5 g L−1 XG on silicon wafers.

whereas the second step may result from the drying step that removes water from the film leading to denser polymer layers and allowing the formation of stable multilayers by intermolecular hydrogen bonds. Several parameters, such as the ChiNC concentration, were investigated in order to obtain ordered nanostructures. The growth process was linear and a regular thickness increment per bilayer was observed. The resulting films showed semireflective properties and developed different colors depending on the thickness. Consequently, this work presents successful LbL assembly of a biobased new coating composed of chitin nanocrystals and xyloglucan involving non electrostatic interactions.



Figure 6. Water contact angles of the (ChiNC-XG)m films at 1.0 g L−1 ChiNC and 0.5 g L−1 XG as a function of the number of layers, m, deposited on a silicon wafer. Empty circles (○) indicate ChiNC deposition and black circles (●) correspond to XG deposition.

ASSOCIATED CONTENT

S Supporting Information *

Data for adsorption of ChiNC and XG on the gold surface registered by the QCM-D sensor as function of time for the three ChiNC concentrations studied (0.5, 1.0, and 3 g L−1), and AFM images of the ChiNC and (ChiNC-XG)8 films at 0.5 and 3 g L−1. This material is available free of charge via the Internet at http://pubs.acs.org.

of ChiNC films could be exploited for new applications, which will be the subject of further investigations.





CONCLUSIONS To the best of our knowledge, this article describes for the first time the interaction between XG and ChiNC surface. Indeed, QCM-D results revealed a complete and irreversible adsorption of XG on ChiNC surface. This fact supported the idea of interaction of XG with polysaccharides surface that have been proposed earlier by molecular modeling in the case of XG/ cellulose adsorption.15,16 This new finding was used to prepare multilayer thin films consisting of alternated ChiNC and XG layers using the spin-assisted LbL procedure. However, for the multilayer fabrication, after adsorption of XG in water, the films were dried during the spinning step. We recently proposed that the drying step induces the formation of hydrogen bonds by the removal of water, which is mandatory for the stabilization of the films. To summarize, the film growth occurs according to two steps: the first one that occurs may involve a combination of electrostatic, van der Waals interactions and hydrogen bonds,

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS A.V. gratefully thanks the financial support of AgreenSkills Postdoctoral Fellowship Program. This work has been also funded by the local council program MATIERES. The authors acknowledge the BIBS platform of INRA Angers-Nantes for the access to microscopy facilities. Emilie Perrin and Nadège Beury are acknowledged for the excellent technical support for the ChiNC preparation and characterization and AFM images, respectively. 193

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dx.doi.org/10.1021/bm401474c | Biomacromolecules 2014, 15, 188−194

Chitin nanocrystal-xyloglucan multilayer thin films.

For the first time, the adsorption of xyloglucan (XG) on chitin nanocrystals (ChiNC) surface was proved using quartz crystal microbalance with dissipa...
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