Rebecca Blell

Xiaofeng Lin

Tom Lindström

Mikael Ankerfors

Matthias Pauly

Olivier Felix

Gero Decher

Generating In-Plane Orientational Order in Multilayer Films Prepared by Spray-Assisted Layer-by-Layer Assembly

Keywords: polyelectrolyte multilayers, layer-by-layer assembly, cellulose nanofibrils, spray-assisted alignment, in-plane orientation, anisotropic materials

We present a simple yet efficient method for orienting cellulose nanofibrils in layer-by-layer assembled films through spray-assisted alignment. While spraying at 90° against a receiving surface produces films with homogeneous in-plane orientation, spraying at smaller angles causes a macroscopic directional surface flow of liquid on the receiving surface and leads to films with substantial in plane anisotropy when nanoscale objects with anisotropic shapes are used as components. First results with cellulose nanofibrils demonstrate that such fibrils are easily aligned by grazing incidence spraying to yield optically birefringent films over large surface areas. We show that the cellulosic nanofibrils are oriented parallel to the spraying direction and that the orientational order depends for example on the distance of the receiving surface from the spray nozzle. The alignment of the nanofibrils and the in-plane anisotropy of the films were independently confirmed by atomic force microscopy, optical microscopy between crossed polarizers, and the ellipsometric determination of the apparent refractive index of the film as a function of the in-plane rotation of the sample with respect to the plane of incidence of the ellipsometer.

The controlled alignment of molecules and nanoscale objects is one key prerequisite in materials science when fabricating materials and devices with anisotropic physical properties. Anisotropic properties refer to a material's physical properties (for example absorbance, refractive index, conductivity, and tensile strength) for the case in which these properties differ when measured along different directions in space. Many chemical components with an anisotropic shape (polymers, liquid crystals, fibrils, metallic nanowires, etc.) have already been aligned in bulk or on surfaces (for example on Teflon surfaces 1 ), including components such as DNA 2 or carbon nanotubes. [START_REF] De Heer | A Carbon Nanotube Field-Emission Electron Source[END_REF] There are many more reports in the literature than can meaningfully be cited here; a selection of reviews [START_REF] Kwiat | Large-Scale Ordered 1D-Nanomaterials Arrays: Assembly or not?[END_REF][START_REF] Liu | Macroscopic-Scale Assembled Nanowire Thin Films and Their Functionalities[END_REF][START_REF] Mei | Recent Advances in Directed Assembly of Nanowires or Nanotubes[END_REF][START_REF] Long | Recent Advances in Large-Scale Assembly of Semiconducting Inorganic Nanowires and Nanofibers for Electronics, Sensors and Photovoltaics[END_REF][START_REF] Ma | The Production of Horizontally Aligned Single-Walled Carbon Nanotubes[END_REF][START_REF] Jiang | Controlled Growth and Assembly of One-Dimensional Ordered Nanostructures of Organic Functional Materials[END_REF][START_REF] Wang | Directed Assembly of Nanowires[END_REF] may serve as a starting point for readers outside the field. Oriented thin films of organic, polymeric, or hybrid materials are used for many applications ranging from optical polarizers to photovoltaic devices or even to cell culture. Orientation in thin films is typically brought about by external fields (mechanical, electrical, or magnetic) or by epitactic growth (templating). Simply dipping of a substrate through Langmuir-Blodgett (LB) monolayers can lead to an alignment of molecules in the dipping direction; see for example ref 11. Another popular method for obtaining oriented thin films consists in simply stretching a plastic foil or a film filled with anisotropic particles. "Rubbing" is another frequently used method for aligning molecules on surfaces, especially for orienting liquid crystals in displays. Table 1 outlines the major principles for inducing orientational order and the usefulness of the different methods in materials science in a very general way. Since highly oriented states are rarely equilibrium states, at least on the macroscopic scale, almost all alignment methods require a matrix material (1D, 2D, or 3D), which allows for local movements during the alignment and which prevents relaxation afterward. Therefore, almost all methods that rely on external fields that are switched off after the alignment have poor permanence unless using a physically or chemically crosslinkable matrix (which imposes restrictions with respect to certain applications). With the exception of inducing orientation by mechanical stretching or surface "wiping" or "rubbing", all of the other methods are often hampered by the limited surface areas over which the respective alignment procedure can be applied (e.g., spin coating). "Wiping" and "rubbing" work very well for orienting liquid crystals, but are prone to induce groove formation on the surface or other types of ablation and both are, as liquid-based shearing, difficult to apply to nonplanar surfaces. Electrical fields require the positioning of electrodes, and magnetic fields require bulky equipment very close to the area to be oriented. Stretching is restricted to surfaces that can be deformed, and they thus do not maintain their original shape. Epitactic orientation is mostly just a transfer of order relying on an existing organization of matter or on patterning of the surface to be templated from and requires an oriented surface to begin with. Note that Table 1 underlines the need for developing additional methods for preparing anisotropic materials with complex composition (multimaterials) and with complex anisotropies (other than unidirectional alignment) over large surface areas. We believe that only bottom-up self-assembly methods will be suitable for preparing macroscale materials and devices with complex composition and complex anisotropy. A particularly successful method for the functionalization of surfaces and the preparation of nanoscale hybrid films is the layer-by-layer (LbL) assembly technique developed by our team since the 1990s. [START_REF] Sukhorukov | Assembly of Polyelectrolyte Multilayer Films by Consecutively Alternating Adsorption of Polynucleotides and Polycations[END_REF][START_REF] Decher | Fuzzy Nanoassemblies: Toward Layered Polymeric Multicomposites[END_REF][START_REF] Seyrek | Layer-by-Layer Assembly of Multifunctional Hybrid Materials and Nanoscale Devices[END_REF][START_REF] Decher | Multilayer Thin Films: Sequential Assembly of Nanocomposite Materials[END_REF] The versatility of this approach arises from the large choice of components that can be assembled on almost any solvent-accessible interface, from its ease of application, even on large surfaces, from its nanoscale precision, and from its very good reproducibility. LbL assembly is usually performed in aqueous media and typically involves the consecutively alternating adsorption of oppositely charged molecules such as polyelectrolytes or objects such as nanoparticles. Overcompensation of the surface charge accompanied by counterion release after each polyion deposition is the major driving force for the multilayer buildup. In a variation of the deposition by adsorption from solution ("dipping"), the sprayassisted assembly of multilayers was introduced by L. Winterton [START_REF] Winterton | Coating of Polymers[END_REF] and J. B. Schlenoff. [START_REF] Schlenoff | Sprayed Polyelectrolyte Multilayers[END_REF] It has the enormous advantage of substantially shortening the film deposition times [START_REF] Izquierdo | Dipping versus Spraying: Exploring the Deposition Conditions for Speeding Up Layer-by-Layer Assembly[END_REF] while generally maintaining the nanostratification of such spray-assembled films. LbL assembly has already been successfully used for the coplanar alignment of clay platelets and nanosheets. [START_REF] Sukhorukov | Assembly of Polyelectrolyte Multilayer Films by Consecutively Alternating Adsorption of Polynucleotides and Polycations[END_REF][START_REF] Kleinfeld | Stepwise Formation of Multilayered Nanostructural Films from Macromolecular Precursors[END_REF][START_REF] Sasaki | Layer-by-Layer Assembly of Titania Nanosheet/Polycation Composite Films[END_REF][START_REF] Malwitz | Orientation of Platelets in Multilayered Nanocomposite Polymer Films[END_REF][START_REF] Priolo | Super Gas Barrier of Transparent Polymer-Clay Multilayer Ultrathin Films[END_REF][START_REF] Tang | Nanostructured Artificial Nacre[END_REF][START_REF] Podsiadlo | Ultrastrong and Stiff Layered Polymer Nanocomposites[END_REF] It seems that, despite its success, the preparation of multilayer films with in-plane order is quite difficult and has been attempted by only a few teams. [START_REF] Schneider | Self-Assembled Monolayers and Multilayered Stacks of Lyotropic Chromonic Liquid Crystalline Dyes with In-Plane Orientational Order[END_REF][START_REF] Ziegler | Photoorientation of Azobenzene Moieties in Self-Assembled Polyelectrolyte Multilayers[END_REF][START_REF] Park | In-Plane Photoalignment of Liquid Crystals by Azobenzene-Polyelectrolyte Layer-by-Layer Ultrathin Films[END_REF][START_REF] Muller | Induced Orientation of r-Helical Polypeptides in Polyelectrolyte Multilayers[END_REF][START_REF] Shim | Single-Walled Carbon Nanotube Combing during Layer-by-Layer Assembly: From Random Adsorption to Aligned Composites[END_REF][START_REF] Gunawidjaja | Buckling Behavior of Highly Oriented Silver Nanowires Encapsulated within Layer-by-Layer Films[END_REF][START_REF] Jiravanichanun | Fabrication of Conjugated Polymer Hybrid Thin Films with Radially Oriented Aluminosilicate Nanofibers by Spin-Assembly[END_REF][START_REF] Koetse | Influence of Charge Density and Distribution on the Internal Structure of Electrostatically Self-assembled Polyelectrolyte Films[END_REF][START_REF] Nogueira | Layer-by-Layer Deposited Chitosan/Silk Fibroin Thin Films with Anisotropic Nanofiber Alignment[END_REF][START_REF] Weitzel | Aggregation and Its Influence on Macroscopic In-Plane Organization in Thin Films of Electrostatically Self-Assembled Perylene-Diimide/Polyelectrolyte Nanofibers[END_REF] Despite the fact that layer-by-layer assembly has the advantages of (a) being open toward a large number of different components that can be used and (b) that in the majority of cases the components are trapped where they arrive on the surface, none of the attempts to achieve in-plane orientation in polyelectrolyte multilayers and related composites have proven to be a general approach for controlling the alignment of anisotropic objects, especially for a large number of layers over large surface areas.

Figure 1. (a) Schematically depiction of a spray jet in which the central axis of the spray cone is parallel to the surface normal.

As a consequence there is almost no macroscopically directed shear flow induced when spraying at a 90° angle against the receiving surface, especially when using small droplet speeds and when spraying from large distances. Note that the nozzle, droplets, and thickness of the liquid film on the surface are not to scale. (b) Grazing incidence spraying. The angle between the center of the spray jet and the surface is typically less than 20° (red lines). Spraying at this geometry produces a liquid flow on the surface in the spraying direction (away from the nozzle). (c) Buildup of LbL-assembled films composed of anionic CNFs and chitosan using the spraying geometries as depicted in (a) (black circles) and (b) (blue squares and red diamonds). The concentrations used for spraying were 2.5 mg/mL for PEI and 1.0 mg/mL for chitosan and CNF. Chitosan was deposited from 0.15 M NaCl; the other compounds were deposited from pure water. All data were obtained using the same nozzle at a liquid flow of 1 mL/min. The black and blue data points were obtained at an air flow of 20 L/min, and the red diamonds at an air flow of 30 L/min. From the many methods of aligning nanorods, nanowires, or nanofibrils on surfaces, we have selected shear flow as the best suited because it requires less bulky equipment than needed for creating strong electrical or magnetic fields. Hydrodynamic alignment is already being carried out at the air/water interface, [START_REF] Sanchez-Iglesias | Highly Transparent and Conductive Films of Densely Aligned Ultrathin Au Nanowire Monolayers[END_REF][START_REF] Tao | Langmuir-Blodgettry of Nanocrystals and Nanowires[END_REF] by "combing" [START_REF] Yao | A Nanoscale Combing Technique for the Large-Scale Assembly of Highly Aligned Nanowires[END_REF] or in capillaries, [START_REF] Håkansson | Hydrodynamic Alignment and Assembly of Nanofibrils Resulting in Strong Cellulose Filaments[END_REF] especially the latter case being a problem for scaling-up to large surface areas. We have therefore begun to induce shear flow on surfaces by grazing incidence spraying (GIS). [START_REF] Decher | Preparation of Coatings Containing at Least One In-Plane Oriented Layer of Anisotropic Shaped Objects[END_REF] While it is somewhat related to "blow alignment", [START_REF] Kim | Fabrication of Nanowire Channels with Unidirectional Alignment and Controlled Length by a Simple, Gas-Blowing-Assisted, Selective-Transfer-Printing Technique[END_REF] "spray alignment" is more versatile because of the better control over physical parameters such as droplet size, droplet speed, and nozzle shape. However, we found only a single publication in the literature [START_REF] Assad | Spray-Coating Route for Highly Aligned and Large-Scale Arrays of Nanowires[END_REF] reporting on a spray-based process, in which highly diluted silicon nanowires are oriented and pinned during droplet evaporation on the receiving hot (!) surface. Note that the requirement for heating the receiving surface makes this method unsuitable for being combined with LbL assembly. Since we must induce orientation at the liquid/solid interface at well-defined temperatures in the range of about 10 to 50 °C and preferentially at ambient temperatures-otherwise the LbL process cannot be controlled well-we have started to develop a true "spray alignment" without evaporating the impinging droplets. [START_REF] Decher | Preparation of Coatings Containing at Least One In-Plane Oriented Layer of Anisotropic Shaped Objects[END_REF] Most importantly, grazing incidence spraying also allows scaling up to large area coatings by moving the spray nozzle with respect to the surface to be coated. [START_REF] Decher | Preparation of Coatings Containing at Least One In-Plane Oriented Layer of Anisotropic Shaped Objects[END_REF] Another advantage of spray-assisted LbL assembly [START_REF] Izquierdo | Dipping versus Spraying: Exploring the Deposition Conditions for Speeding Up Layer-by-Layer Assembly[END_REF] is that it can be used for preparing well-organized multicomponent stacks, which has not been demonstrated for any of the other alignment methods described above. [START_REF] Izquierdo | Dipping versus Spraying: Exploring the Deposition Conditions for Speeding Up Layer-by-Layer Assembly[END_REF][START_REF] Sanchez-Iglesias | Highly Transparent and Conductive Films of Densely Aligned Ultrathin Au Nanowire Monolayers[END_REF][START_REF] Tao | Langmuir-Blodgettry of Nanocrystals and Nanowires[END_REF][START_REF] Yao | A Nanoscale Combing Technique for the Large-Scale Assembly of Highly Aligned Nanowires[END_REF][START_REF] Håkansson | Hydrodynamic Alignment and Assembly of Nanofibrils Resulting in Strong Cellulose Filaments[END_REF][START_REF] Decher | Preparation of Coatings Containing at Least One In-Plane Oriented Layer of Anisotropic Shaped Objects[END_REF][START_REF] Kim | Fabrication of Nanowire Channels with Unidirectional Alignment and Controlled Length by a Simple, Gas-Blowing-Assisted, Selective-Transfer-Printing Technique[END_REF][START_REF] Assad | Spray-Coating Route for Highly Aligned and Large-Scale Arrays of Nanowires[END_REF] Cellulosic nanofibers (CNFs) are cheap and readily available nanoscale objects with large aspect ratios possessing diameters of a few nanometers and lengths about 250 to 500 nm. Recently both cellulose nanocrystals (CNCs) and CNFs became available in anionic and cationic form and therefore have been used to prepare LbL films. [START_REF] Wågberg | The Build-Up of Polyelectrolyte Multilayers of Microfibrillated Cellulose and Cationic Polyelectrolytes[END_REF][START_REF] Utsel | Thermoresponsive Nanocomposites from Multilayers of Nanofibrillated Cellulose and Specially Designed N-Isopropylacrylamide Based Polymers[END_REF] CNCs have already been oriented by electric fields, magnetic fields, and shear force both in thin films and in suspensions. [START_REF] Bordel | Orientation of Native Cellulose in an Electric Field[END_REF][START_REF] Cranston | Formation of Cellulose-Based Electrostatic Layer-by-Layer Films in a Magnetic Field[END_REF][START_REF] Sugiyama | Orientation of Cellulose Microcrystals by Strong Magnetic Fields[END_REF][START_REF] Kimura | Magnetic Alignment of the Chiral Nematic Phase of a Cellulose Microfibril Suspension[END_REF][START_REF] Frka-Petesic | Negative Diamagnetic Anisotropy and Birefringence of Cellulose Nanocrystals[END_REF][START_REF] Ebeling | Shear-Induced Orientation Phenomena in Suspensions of Cellulose Microcrystals, Revealed by Small Angle X-ray Scattering[END_REF][START_REF] Heux | Nonflocculating and Chiral-Nematic Self-ordering of Cellulose Microcrystals Suspensions in Nonpolar Solvents[END_REF] Further, the stretching of freestanding LbL films of CNCs has also led to aligned systems. Very recently K. Håkansson et al. reported the hydrodynamic alignment of CNFs in glass capillaries yielding strong cellulose filaments. 39

Figure 3. (a) Schematic depiction of the intersection of the spray jet with the receiving surface (side view). The bluish oval indicates the area (1-1.5 cm 2 ) in which the nanofibrils have the highest orientation. (b) Top view of the spray cone arriving on the receiving surface. Note that the border of the bluish oval is in reality much more fuzzy on the right (downstream side) than on the left since the liquid flow across the surface decreases from left

to right, where it gradually becomes zero. The red spots (1-4) indicate the locations where the AFM images were taken, whose slightly smoothed Fourier transforms are shown on the right (c). The approximate major axis is depicted in green, and the approximate minor axis is depicted in blue. AFM images were taken on a LbL film composed of one layer pair of PEI/aCNF. The concentrations used for spraying were 2.5 mg/mL for PEI and 2.0 mg/mL for CNF. Both compounds were deposited from pure water.

Here we report on the assembly of oriented LbL mono-and multilayer films of anionic cellulose nanofibrils (aCNFs, Scheme 1a) with a polycation such as poly(ethylene imine) (PEI) or chitosan (Scheme 1b) using grazing incidence spraying, [START_REF] Decher | Preparation of Coatings Containing at Least One In-Plane Oriented Layer of Anisotropic Shaped Objects[END_REF] a simple yet efficient method for inducing in-plane alignment of each deposited aCNF layer (Figure 1). The alignment of the nanofibrils and the in-plane anisotropy of the films were determined by atomic force microscopy, optical microscopy between crossed polarizers, and ellipsometry.

RESULTS AND DISCUSSION

Atomic force microscopy (AFM) topographies of a submonolayer of aCNFs on a PEI layer (Figure S1) show that most of the aCNFs incorporated in the LbL films are around 2 nm wide and about 250 to 500 nm long. They are also mostly individual fibrils freed from the bundles during ultrasonication. [START_REF] Wågberg | The Build-Up of Polyelectrolyte Multilayers of Microfibrillated Cellulose and Cationic Polyelectrolytes[END_REF] aCNF LbL multilayers were prepared both by dipping [START_REF] Decher | Fuzzy Nanoassemblies: Toward Layered Polymeric Multicomposites[END_REF] and by sprayassisted LbL assembly [START_REF] Schlenoff | Sprayed Polyelectrolyte Multilayers[END_REF][START_REF] Izquierdo | Dipping versus Spraying: Exploring the Deposition Conditions for Speeding Up Layer-by-Layer Assembly[END_REF] using two different spraying geometries: classic spraying (Figure 1a) and grazing incidence spraying (Figure 1b). In the classical method, solutions are sprayed horizontally and perpendicular to the receiving surface, while the grazing incidence spraying method is performed using a high-speed spray jet hitting the surface nearly parallel to the receiving surface at an angle of typically less than 20°. The aCNF suspension is sprayed onto the receiving surface from a distance of around 1 cm from the nozzle. Grazing incidence spraying, previously also named low-angle spraying or spraying at offnormal angles, has been reported in jet washing devices [START_REF] Gill | Low Angle Spraying Machine[END_REF][START_REF] Grant | Spray/Jet Washing Device[END_REF] and in systems for precision cleaning [START_REF] Peterson | System for Precision Cleaning by Jet Spray[END_REF] and has also been studied in thermal and cold spraying [START_REF] Sobolev | Effect of Droplet Impact Angle on Flattening of Splat in Thermal Spraying[END_REF][START_REF] Li | Effect of Different Incidence Angles on Bonding Performance in Cold Spraying[END_REF] but has, to the best of our knowledge, never been used for preparing LbL-assembled films composed of oriented nanoobjects. A huge advantage of grazing incidence spraying is that, unlike, for example, spincoating, it can be applied to surfaces of different size and even different shape and that the axis of the spray jet determines the direction of the orientation. Figure 1c shows that the film growth is quite regular for orthogonal spraying (Figure 1a) and for grazing incidence spraying (Figure 1b). Please note that ellipsometrically determined film thicknesses of optically anisotropic materials do not represent absolute values since the refractive index of such films varies with the in-plane direction. However, in our case the deviation from the absolute film thickness is quite small since the refractive index varies only between about 1.48 and about 1.57 (see below). It seems therefore safe to infer from Figure 1c that LbL films of anionic CNFs obtained by regular spraying are much thinner than those obtained by GIS. Such differences of the film thickness of LbL-assembled multilayers prepared from identical components are not surprising. It is well established that small changes of the preparation conditions may have substantial influence on the film thickness and on other structural characteristics (roughness, density, etc.). Several methods were used for determining the alignment of the CNFs in LbL-assembled films. The surface morphology of nonaligned and aligned CNF films was studied on a single aCNF layer only because this yields the sharpest images. Figure 2 shows the AFM images of dipped (Figure 2a), orthogonally sprayed (Figure 2b), and GIS (Figure 2c) films. As already seen by the naked eye on the AFM images, Figure 2a andb show no preferential orientation of the cellulosic fibrils, whereas the fibrils in Figure 2c are clearly oriented in one in-plane direction. Note that the direction of alignment (top-to-bottom in Figure 2c) coincides with the spraying direction. This first impression was further corroborated by taking the fast Fourier transforms (FFTs) of the AFM images. The FFTs of dipped (Figure 2d) and orthogonally sprayed (Figure 2e) films have an almost circular shape, whereas the FFT of the GIS sample (Figure 2f) has an ellipsoidal shape. The AFM images have then been analyzed using the plugin OrientationJ 57 developed for ImageJ, [START_REF] Schneider | NIH Image to ImageJ: 25 Years of Image Analysis[END_REF][START_REF] Abramoff | Image Processing with ImageJ[END_REF] which is based on the analysis of the structure tensor in a local neighborhood. An orientation direction is determined for each pixel of the image, and this local orientation is color-coded (different colors represent different alignment directions) in order to obtain the map of orientation for the dipped (Figure 2g), orthogonally sprayed (Figure 2h), and GIS samples (Figure 2i). It appears clearly that only the GIS sample is oriented, as indicated by the predominance of the red color, which codes for an orientation in the vertical direction. The distribution of orientation can be extracted, and a 2D order parameter (S2D) can be calculated (eq 1) and be used to characterize the degree of alignment.

S2D = ⟨2 cos 2 θ -1⟩ ( 1
)
where θ is the angle between each nanofibril principal axis and the spraying direction. The 2D order parameter S2D can take values between 0 and 1, where S2D = 0 corresponds to a random distribution of CNFs (isotropic films) and S2D = 1 corresponds to a perfectly parallel alignment (θ = 0°, anisotropic films). Correspondingly, the distribution of orientation is almost flat for the dipped (Figure 2j) and orthogonally sprayed (Figure 2k) samples, which is also illustrated by their low 2D order parameter (respectively S2D = 0.04 and S2D = 0.08). In contrast, the angular distribution of the nanofibrils in the GIS assembled films (Figure 2l) shows a sharp peak, with a high 2D order parameter (S2D = 0.82), which corresponds to 90% of the CNFs oriented within ±30° and 50% oriented within ±10°.

The orientation of the fibrils depends strongly on the spraying conditions (direction and inclination of the spray jet (Figure S2); size, speed, and density of the spray droplets).

Exemplarily we show the dependence of the alignment with respect to the position of the sample area on the position within the spray jet. Figure 3 shows four FFTs that were obtained at the positions indicated in the drawing showing the spraying geometry (Figure 3b). Note that different positions imply different trajectory directions of the spray droplets and different trajectory lengths from the nozzle to the contact point of the droplet with the liquid film on top of the receiving surface. In general longer trajectory lengths correspond to longer flying times of the spray droplets and therefore smaller droplet speeds. It seems reasonable to assume that in a somewhat divergent liquid flow on a surface the induced liquid shear decreases with longer trajectory lengths and therefore causes a reduced orientation with increasing distance from the nozzle (Figure S3). With short trajectories as shown in Figure 3a (approximately 1 cm), we expect only negligible effects from the reduction of droplet size due to water evaporation during flight and from the corresponding reduction in temperature.

Since effects from droplet size and temperature cannot be ruled out in principle in the case of CNFs and other gel-forming compounds, we worked at CNF concentrations sufficiently below the gelling concentration of CNFs of about 2 wt %. Note that the FFT taken at position 4 is almost circular because this position is out of the bounds of the spray jet and the spray droplets arriving at this position are transported there by random air flow caused by turbulences. Table 2 gives for each spot the values of the orientation index of the fibrils as derived from the FFT axis lengths and the values of the 2D order parameter. The orientationcolor-coded images and the corresponding order parameters are given in detail in Figure S4. Data obtained by analysis of the orientation distribution of individual fibers corroborate what was seen by FFT for the ensemble. When spraying with nonmoving nozzles, the homogeneous region of the CNF monolayer in the center of the arriving spray jet (Figure 3a) in which all fibrils have the same direction of alignment has a size of about 1 to 1.5 cm 2 .

Table 2. Orientation Index and 2D Orientation Parameter for Points 1, 2, 3, and 4 of Figure 3 a a The orientation index is defined as 1 -(S/L), with S and L being the length of the major axis and of the minor axis of the ellipses on the corresponding FFT images. FFT images were obtained from AFM images of one layer pair of PEI/aCNF. The concentrations used for spraying were 2.5 mg/mL for PEI and 2.0 mg/mL for CNF. Both compounds were deposited from pure water.

After establishing in which area of the spray jet arriving on a static receiving surface a welloriented monolayer of aCNFs is formed, it was interesting to find out if it is possible to maintain the monolayer alignment throughout an entire multilayer film. For this purpose (aCNF/chitosan)m multilayers were built on surfaces primed with a first layer of poly(ethylene imine), the cellulosic nanofibrils were assembled by grazing incidence spraying, and the chitosan was deposited by orthogonal spraying. The number of layer pairs was varied between 1 and 120. Since aCNFs do not possess unique chromophores whose polarized absorbance can be associated with the long axis of the fibrils, we estimated the in-plane variation of the refractive index (Figure 4a), and we probed the linear birefringence (Figure 4b) of these oriented films in order to demonstrate the alignment. While the accurate ellipsometric determination of the optical constants of anisotropic materials requires dedicated instruments (generalized ellipsometry or Müller ellipsometry), [START_REF] Garcia-Caurel | Application of Spectroscopic Ellipsometry and Mueller Ellipsometry to Optical Characterization[END_REF] we have probed the anisotropic character of the optical properties of our films with a routine ellipsometer by measuring the shape of the in-plane distribution of an apparent refractive index. For this purpose the anisotropic (aCNF/chitosan)m film was rotated around the laser spot of the ellipsometer at 10 degree intervals, and an apparent refractive index n of the film was determined while imposing a constant value for the film thickness d in the derivation of n and d from the ellipsometric angles psi (Ψ) and delta (Δ) and by using a fitting model in which an isotropic refractive index is used. The results for a spray-aligned sample composed of 80 layer pairs are shown in Figure 4a, where the gray arrow in the polar plot indicates the spraying direction. From the analysis of our AFM data by FFT and by OrientationJ we gather that the cellulosic nanofibrils are predominantly oriented with their long axis parallel to the spraying direction. Combining this information with Figure 4a we find that the smallest apparent refractive index (about 1.48) is observed parallel to the alignment direction and that the largest apparent refractive index (about 1.57) is observed perpendicular to the alignment direction of the fibrils. The refractive index of a nonaligned LbL film of CNFs is about 1.52, which falls almost in the middle between the maximum and minimum apparent refractive indices of aligned films. These results obtained by ellipsometry are somewhat counterintuitive, as the refractive index along the long axis of the fibrils is often expected to be larger than the refractive index perpendicular to the fibrils. [START_REF] Cranston | Birefringence in Spin-Coated Films Containing Cellulose Nanocrystals[END_REF][START_REF]Polymer Handbook[END_REF] Unfortunately, at present the measurements on oriented cellulose containing composite systems are controversial [START_REF] Cranston | Birefringence in Spin-Coated Films Containing Cellulose Nanocrystals[END_REF][START_REF] Songsurang | Optical Anisotropy in Solution-Cast Film of Cellulose Triacetate[END_REF] and cannot be used as a simple guidance for arguing about the alignment direction of the respective cellulosic species. However, the refractive index is a materials property that is independent of the thickness of the film, that is, the number of deposited layer pairs. In contrast, optical microscopy with crossed polarizers allows determining the (relative) retardation (or the optical path difference), which is related to the linear birefringence multiplied by the thickness of the medium and can therefore be used to demonstrate that the whole film possesses an anisotropic structure and not just the first layers. The intensity changes of the reflected light are thus a function of the in-plane rotation and the thickness of the film and easily recorded as relative brightness with a digital camera. Figure 4b shows the in-plane variation of the relative brightness of different multilayer films as obtained from optical microscopy using crossed polarizers. As expected, films obtained by orthogonal spraying (or by dipping) show only negligible birefringence (Figure 4b, red data points). For anisotropic films a clear dependence of the relative brightness on the number of deposited layer pairs is observed: the thicker the films, the more intense the relative brightness gets (Figure S5). Figure 4b shows the in-plane relative brightness of a spray-aligned multilayer film composed of 80 layer pairs (blue data points). What is interesting in this case is that the shape of the dispersion does not -on first sight-correspond to the expectations for a unidirectional alignment of the CNFs. While for Figure 4b a C4 symmetry would be expected, a C2 symmetry is observed. A rotation of the sample to the right by a given angle does not lead to the same reflected intensity as a rotation of the sample around the same angle to the left, as observed on the photographic images of a sample between crossed polarizers (Figure S6). The three images (Figures 4c-e) show the edge region of a multilayer film on a silicon wafer, where the spraying direction is indicated by the gray arrow. We show the edge region of the sample rather than a homogeneously birefringent region so that the rotation of the sample under crossed polarizers is more easily visualized. The bluish areas represent the intensity of the birefringence of the film, and the black areas in the lower part of each image are outside of the silicon wafer. The grayish/blackish stripe going through the center of each image close to the gray arrow is the edge of the silicon wafer and is due to border effects. In Figure 4c-e the sample is rotated in steps of 45° to the right, with Figure 4c corresponding to the direction of 135° in Figure 4b, Figure 4d corresponding to the direction of 180° in Figure 4b, and Figure 4e corresponding to the direction of 225° in Figure 4b. The asymmetry of the relative brightness of the four lobes shown in Figure 4b also reproduces the original photographs 4c and 4e, which are part of the series of images from which the data in 4a were sampled. These two puzzling observations of an asymmetry in the apparent linear birefringence and of the unexpected apparent refractive indices may have their origin in the chirality of the cellulose nanofibrils, which is due to the handedness of cellulose. Note that the in-plane distribution of the reflected intensity of unidirectionally aligned nonchiral objects (for example silver nanowires) is perfectly symmetric. However, if one investigates the birefringence of unidirectionally aligned chiral objects, the matrix elements for circular dichroism and for circular birefringence of the Müller matrix 60 may become nonzero, and this can influence the observed intensities of the relative brightness, which is depicted in Figure 4b, and the apparent refractive indices measured by ellipsometry (Figure 4a). In such a case, for interpreting the response of a medium to linearly polarized light the full Müller matrix must be taken into account and not only the isotropic and linear matrix elements. Since CNFs and in our case also the polycation chitosan are chiral compounds, the terms of the Müller matrix that are linked to circular birefringence should possess a nonzero value, and this could lead to a nonsymmetric angular dispersion of the reflected intensity observed under crossed polarizers. A careful investigation (that goes beyond the scope of the present article) combining generalized ellipsometry with an anisotropic layer model, Müller matrix polarimetry, and optical reflectometry at different angles and wavelengths would be needed for determining more accurately the birefringence and refractive indices of these chiral anisotropic systems.

(c) Maximum relative brightness of the less intense lobe of (b) (inplane angle 135°). (d) Minimum relative brightness of (b) which is observed parallel to the spraying direction (inplane angle 180°). (e) Maximum relative brightness of the more intense lobe of (b) (in-plane angle 225°

). The left part of (d) was digitally brightened to better show that in this orientation of the wafer the reflected intensity of the multilayer film is at its minimum. The brightness observed at the lower border of the silicon wafer (above the gray arrow) is due to edge effects. All lines connecting the data points are a guide to the eye only, and all gray arrows indicate the spraying direction. The concentrations used for spraying were 2.5 mg/mL for PEI and 1.0 mg/mL for chitosan and CNF. Chitosan was deposited from 0.15 M NaCl; the other compounds were deposited from pure water.

CONCLUSIONS

Our results document that highly anisotropic materials can be prepared by using shear alignment during the deposition of layer-by-layer assembled films. In the present work the inplane orientation of cellulose nanofibrils in the spraying direction was demonstrated by analyzing the surface morphologies of LbL monolayers of aCNF on silicon wafers. In LbL multilayers the apparent refractive index of the film has a minimum value in the alignment direction and a maximum value orthogonal to the alignment direction. Unlike the refractive index, the relative brightness of a birefringent material under crossed polarizers depends on the number of layer pairs constituting the film. This shows that all individual aCNF layers within a multilayer sample contribute to its birefringence and are thus oriented in the same direction. The fact that spray alignment in combination with layer-by-layer assembly leads to highly anisotropic materials makes it possible to prepare such materials over large surface areas. Preliminary experiments on postcard-sized surfaces are already pointing to the fact that even larger surfaces can be targeted. The use of LbL assembly permits the use of different chemical compounds in different layers, which enables the preparation of multimaterials with multiple engineered properties. The fact that grazing incidence spraying easily allows choosing the alignment direction in each individual layer makes it possible to prepare materials with more complex anisotropies than the unidirectional alignment reported here.

METHODS

Materials.

The anionic cellulose nanofibrils (aCNF bearing carboxymethyl functional groups, degree of substitution of around 0.1 determined using conductometric titration64 of the pulp before homogenization) were obtained from Innventia AB (Stockholm, Sweden) as a pulp containing 2% fibers in water. The aCNF pulp used as stock sample was produced by means of carboxymethylation of a never-dried softwood dissolving pulp (trade name: Dissolving Plus; Domsjö Mill, Domsjö Fabriker, Sweden) followed by high-pressure homogenization using a Microfluidizer M-110EH (Microfluidics Corp., USA). Detailed procedures for the pulp production have earlier been described by Wågberg et al. [START_REF] Wågberg | The Build-Up of Polyelectrolyte Multilayers of Microfibrillated Cellulose and Cationic Polyelectrolytes[END_REF] One gram of aCNF stock sample (2 wt %) was mechanically dispersed in 10 mL of Milli-Q water (resistivity of 18.2 MΩ•cm, Milli-Q Gradient system, Millipore, Molsheim, France) and sonicated with a tip sonicator (Vibra Cell 75042 from Bioblock Scientific, IIlkirch, France) for 30 min at 20% amplitude. The resulting suspension was centrifuged for 4 h at 18 000 rpm (centrifuge 4K10 with Rotor Nr 12254 from Sigma, Lyon, France), and the resulting supernatant was used as deposition solution. This procedure allows the preparation of a 0.2 wt % aCNF suspension used for the monolayer study. A 0.1 wt % aCNF suspension was used for multilayer thin films, which were prepared by mixing 1 g of aCNF stock sample (2 wt %) with 20 mL of Milli-Q water followed by sonication and centrifugation as explained above.

Poly(ethylene imine) (trade name Lupasol WF, PEI, Mw ≈ 25 000 g/mol) was freely provided by BASF (Ludwigshafen, Germany) and dialyzed using Zellutrans regenerated cellulose tubular membrane from Carl Roth GmbH with a molecular weight cutoff of 14 000 g/mol followed by freeze-drying. A PEI solution at 2.5 mg/mL was prepared using Milli-Q water. No pH adjustment of the solution was done. Chitosan (medium molecular weight) was purchased from Sigma-Aldrich (Lyon, France) and used as received. Chitosan solution at 1 mg/mL was prepared by first dispersing the required chitosan mass in a few milliliters of acetic acid and then by adding a 0.15 M NaCl solution.

Substrate Preparation. The silicon wafers (Si wafers, WaferNet, Inc., San José, CA, USA) were initially rinsed extensively with acetone and then with Milli-Q water, blown dried with compressed air, and finally plasma treated for 3 min in a plasma cleaner (Harrick Plasma, Ithaca, NY, USA) at medium intensity.

LbL Deposition. LbL films were prepared either by dipping, orthogonal spraying-assisted assembly, or grazing incidence spraying. All films were deposited on a substrate coated with a precursor layer of PEI. For LbL films formed by dipping, the PEI-modified substrate was dipped for 20 min in the aCNF suspension followed by three rinsing steps of 2 min in Milli-Q water. The substrate was then dried with compressed air. The same steps were applied for the deposition of chitosan. This procedure consisted of one deposition cycle (layer pair) and was repeated until the desired thickness was reached. The preparation of thick (aCNF/chitosan)m multilayers was performed using a homemade dipping robot, consisting of three motorized arms (x, y, z directions), a drying station, an interface from ISEL (Houdan, France), and a Labview homemade program.

The deposition was done according to the procedure described above.

The orthogonal spraying-assisted deposition of aCNF and chitosan was carried out using an air pump spray can made of poly(ethylene) and poly(propylene) purchased from Carl Roth GmH (Germany). Each can was filled with the corresponding liquid and pressurized by pumping cycles. Spraying was carried out perpendicularly to the receiving surface, which was fixed in a vertical orientation. Each solution was sprayed for 10 s onto the surface, followed by a rinsing step with Milli-Q water for 20 s. Prior to ellipsometric measurements, films were dried with compressed air.

For GIS deposition, an aqueous suspension of aCNF was deposited on a PEI-coated silicon wafer for 10 s using a homemade spraying system. The latter includes gas flow controllers to adjust the air flow rate (model Red-Y, Voegtlin, Aesch, Switzerland), liquid handling pumps (model M50, VICI, Schenkon, Switzerland), and two-fluid nozzles (internal diameter: 300 μm, Spraying Systems, Wheaton, IL, USA). The nozzle was placed at a distance of 1 cm from the substrate. The liquid flow rate was set to 1 mL/min, and the air flow to 47 L/min. The angle between the spray cone main axis and the receiver substrate was below 5°. Chitosan was deposited by orthogonal spraying using an air pump spray can. The deposition of aCNF or chitosan was followed by a rinsing step with Milli-Q water for 20 s using an air pump spray can. Finally, the substrate was dried using air flow.

Ellipsometry. Thickness measurements of LbL films were performed using either a PLASMOS SD 2300 operating at a wavelength of 632.8 nm and with an angle of 70° or a SENpro spectroscopic ellipsometer (SENTECH Instruments GmbH, Berlin, Germany) operating between 300 and 1050 nm and with an angle of 70°. For thickness measurements, the refractive index was set at n = 1.465 and assumed to be constant. Each data point is an average of 10 measurements at random positions on the wafer. This procedure leads to slightly inexact absolute thickness values, but it allows the determination of the thickness and sufficient precision for the comparison of the buildup and homogeneity of the different films. The variation of apparent refractive index for anisotropic samples as a function of aCNF orientation was measured using the SENpro spectroscopic ellipsometer. Therefore, anisotropic samples were rotated by 10° steps in the plane parallel to the substrate surface prior to measurements, and the ellipsometric angles psi (Ψ) and delta (Δ) were fitted at a fixed film thickness of 245 nm with isotropic refractive indices.

Atomic Force Microscopy. Tapping mode atomic force microscopy was performed on an AFM Multimode from Bruker Nano Surface (Palaiseau, France) with a Nanoscope IV controller from Veeco (Mannheim, Germany) and noncoated silicon cantilevers (resonance frequency 300 kHz, resonance constant of 40 N/m, and radius below 10 nm). Phase and height modes were recorded simultaneously using a constant scan rate of 1.3 Hz with a resolution of 512 × 512 pixels. Orientation of aCNF. The orientation analysis of AFM images was carried out using the ImageJ software. The fast Fourier transform function of ImageJ was used to convert AFM images from "real" space into mathematically defined frequency space. Resulting FFT output images contain gray-scale pixels that are distributed in a pattern that reflects the degree of alignment present in the original data image. A round FFT pattern indicates no orientation in the original AFM image, while an elongated FFT pattern is characteristic of aCNF orientation in a given direction of the AFM image. In addition, OrientationJ, a plugin developed for ImageJ, was used to determine the distribution of orientation of aCNF with respect to the spraying direction. It is based on the analysis of the local structure tensor in the local neighborhood of each pixel of the original AFM pictures. As a result, an orientation angle is given for each pixel, and a color code that represents each angle by a color is used to build. Finally, the angle distribution can be extracted. The distribution is nonweighted (i.e., 1 pixel = 1 count), but the pixels for which the values of the coherency or the energy are lower than 20% have been ignored. Indeed, these pixels are often artifacts, for instance noisy pixels or located at edges and corners.

Cross-Polarized Light Microscopy. The reflected intensity under polarized white light of anisotropic samples was determined using a Leica DM-RX polarized light microscope in which two polarizers are oriented at 90° from each other. The angle between the aCNF orientation direction and the incident light polarization plane has been varied. Micrographs of each sample have been taken every 5° using a camera and transformed to a 8-bit gray-scale image using ImageJ. The average gray value is measured for each picture and plotted as a function of the angle (Figure S6).

ASSOCIATED CONTENT Supporting Information

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.6b04191. AFM picture of a sub-monolayer of aCNFs and their size distribution, angular dispersion of the in-plane birefringence as a function of the incidence angle, angular dispersion of the in-plane birefringence as a function of the nozzle-substrate distance, description of the AFM image analysis procedure, and angular dispersion of the in-plane birefringence as a function of the layer number.

Scheme 1 .

 1 Scheme 1. Chemical structures of carboxymethylcellulose present in the anionic cellulosic nanofibers (a) and of chitosan that was used as cationic polyelectrolyte (b).

Figure 2 .

 2 Figure 2. AFM images (a, b, c, 2 × 2 μm 2 scans) and corresponding fast Fourier transforms (d, e, f), orientationcolor-coded pictures (g, h, i), and angular distribution (j, k, l) of one layer pair of PEI/aCNF obtained by dipping (a, d, g, j), orthogonal spraying (b, e, h, k), and grazing incidence spraying (c, f, i, l). The inset in picture (g) corresponds to the color code for orientation.

Figure 4 .

 4 Figure 4. (a) Angular variation of the apparent refractive index as determined ellipsometrically using a fixed thickness (d = 245 nm) of the LbL film PEI(aCNF/chitosan)80 and assuming an apparent refractive index. (b) Angular dispersion of the in-plane relative brightness under crossed polarizers. Red data points were obtained from a nonaligned sample; the blue data points are from the same aligned multilayer sample as in (a) and (c)-(e). (c, d, and e) Optical micrographs taken at the edge of a silicon wafer under crossed polarizers rotated -45°, 0°, and 45° with respect to the crossed polarizers. (c) Maximum relative brightness of the less intense lobe of (b) (inplane angle 135°). (d) Minimum relative brightness of (b) which is observed parallel to the spraying direction (inplane angle 180°). (e) Maximum relative brightness of the more intense lobe of (b) (in-plane angle 225°). The left part of (d) was digitally brightened to better show that in this orientation of the wafer the reflected intensity of the multilayer film is at its minimum. The brightness observed at the lower border of the silicon wafer (above the gray arrow) is due to edge effects. All lines connecting the data points are a guide to the eye only, and all gray arrows indicate the spraying direction. The concentrations used for spraying were 2.5 mg/mL for PEI and 1.0 mg/mL for chitosan and CNF. Chitosan was deposited from 0.15 M NaCl; the other compounds were deposited from pure water.

Table 1 .

 1 Comparison of the Level of Anisotropy

a Note that Table1somewhat oversimplifies in generalizing the characteristics of different alignment methods, and there are many exceptions to the above.

ACKNOWLEDGMENTS

We gratefully acknowledge financial support from the European Community (SustainComp project, FP7-NMP-214660) and the Centre National de la Recherche Scientifique (CNRS), France. All authors thank Dr. Martin Brinkmann (ICS, France) for providing access to the optical microscope, Christophe Contal (ICS, France) for the AFM training and imaging, and Dr. Oriol Arteaga (University of Barcelona, Spain) for fruitful discussions. M.P. thanks the "Chaire d'Excellence" program of CNRS and the Université de Strasbourg for partial funding. G.D. thanks the Institut Universitaire de France for financial support.

AUTHOR INFORMATION

Corresponding Author *E-mail: decher@unistra.fr.