A Versatile Method for the Distance-Dependent Structural

Article Cite This: Langmuir XXXX, XXX, XXX-XXX

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A Versatile Method for the Distance-Dependent Structural Characterization of Interacting Soft Interfaces by Neutron Reflectometry Samantha Micciulla,†,‡ Yuri Gerelli,‡ Richard A. Campbell,‡ and Emanuel Schneck*,† †

Max Planck Institute of Colloids and Interfaces, 14476 Potsdam, Germany Institut Laue-Langevin, 38000 Grenoble, France

‡

S Supporting Information *

ABSTRACT: Interactions between soft interfaces govern the behavior of emulsions and foams and crucially influence the functions of biological entities like membranes. To understand the character of these interactions, detailed insight into the interfaces’ structural response in terms of molecular arrangements and conformations is often essential. This requires the realization of controlled interaction conditions and surface-sensitive techniques capable of resolving the structure of buried interfaces. Here, we present a new approach to determine the distance-dependent structure of interacting soft interfaces by neutron reflectometry. A solid/water interface and a water/oil interface are functionalized independently and initially macroscopically separated. They are then brought into contact and structurally characterized under interacting conditions. The nanometric distance between the two interfaces can be varied via the exertion of osmotic pressures. Our first experiments on lipid-anchored polymer brushes interacting across water with solid-grafted polyelectrolyte brushes and with bare silicon surfaces reveal qualitatively different interaction scenarios depending on the chemical composition of the two involved interfaces.

1. INTRODUCTION Soft interfaces consisting of two-dimensional molecular assemblies play important roles in biology and in colloidal and soft matter science.1−3 Cells and tissues are congested with membranes and protein or carbohydrate complexes, whose functions depend on the physical interactions between their surfaces. Interactions involving biological membranes, for instance, govern cell adhesion,4 the properties of bacterial biofilms,5 and the adsorption of organisms to material surfaces.6 Similarly, interactions between technological soft interfaces play important roles in a multitude of applications such as liquid purification, lubrication, and separation chemistry. In this context, liquid/liquid interfaces and foams are often stabilized with self-assembled monolayers of surfactants, proteins, or polymers, which render the interfacial forces predominantly repulsive.7−9 Soft interfaces typically exhibit a close relation between their interaction characteristics, in terms of range and strength, and the spatial organization and conformation of the molecules residing at the interfaces.10 For instance, the interaction between surfaces displaying extended charged molecules depends on the ability of the molecules to rearrange in an electrostatically favorable manner upon surface approach. Similarly, polymer-decorated interfaces interact differently if the polymer chains can interpenetrate. Conversely, molecular conformations and the distributions of ions or solutes can exhibit a considerable response when two interfaces are brought © XXXX American Chemical Society

into contact and molecular exchange between the surfaces can occur.11,12 Knowledge of such structural details of interacting surfaces is thus valuable and sometimes a prerequisite to understand the nature of the interaction. Experimental insight into structures ”buried” between two soft interfaces is difficult to obtain. The use of scanning nearfield techniques is prevented because they cannot reach the interfacial region without perforating one of the surfaces. The nanometric length scales of the relevant structures are furthermore inaccessible to optical far-field techniques. Also, the structural features of interest are sensitive to thermodynamic conditions, rendering all cryo-based techniques inadequate. X-ray and neutron scattering are thus essentially the only techniques that can probe such structures with the required subnanometer resolution. Among them, neutron scattering is truly nondestructive and has the advantage of isotopic contrast variation, where certain chemical components can be highlighted with respect to their surroundings by selective deuteration or the use of heavy water. While specular neutron reflectometry (NR) can unambiguously determine matter density profiles perpendicular to an interface,13−15 it Special Issue: Early Career Authors in Fundamental Colloid and Interface Science Received: August 21, 2017 Revised: October 16, 2017 Published: October 17, 2017 A

DOI: 10.1021/acs.langmuir.7b02971 Langmuir XXXX, XXX, XXX−XXX

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H2O was ultrapure Milli-Q water (18 MΩ cm−1, 23 °C) throughout this study. The phospholipids 1,2-distearoyl-glycero-3-phosphocholine (DSPC), 1,2-distearoyl-d70-glycero-3-phosphocholine (d-DSPC), and 1,2-dipalmitoyl-glycero-3-phospho-L-serine sodium salt (DPPS), as well as the lipopolymer 1,2-distearoyl-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000 ammonium salt (PEGlipid) were purchased from Avanti Lipids (Alabaster, AL). The phospholipid 1,2-distearoyl-glycero-3-phospho-L-serine sodium salt (DSPS) was from Sigma-Aldrich (St. Quentin, France). DSPC, dDSPC, and PEG-lipid were dissolved in chloroform; DSPS and DPPS were dissolved in chloroform:methanol mixtures (2:1 v/v). Phospholipid/lipopolymer mixtures with final concentration 1 mg/mL were prepared by mixing solutions of zwitterionic (DSPC) or charged (DPPS, DSPS) phospholipids with a solution of PEG-lipid in appropriate ratios, so that a PEG-lipid mole fraction of 10% ( f = 0.1) was realized. Water was used without the addition of salt, so that only the counterions associated with charged molecular groups were present in the aqueous phase in all cases. 2.2. Sample Preparation. 2.2.1. Preparation of Lipid-Anchored PEG Brushes at the Air/Water Interface. Lipid-anchored PEG brushes were formed at the air/water interface in Teflon troughs with a surface area of (50 × 120) mm2 by spreading defined amounts of phospholipid/PEG-lipid solutions. The amount of solution was calculated on the basis of Langmuir isotherms as shown in Figure S2 of the Supporting Information. Monolayers containing DSPC or DPPS matrix were prepared at a surface pressure of π ≈ 45 mN/m, while monolayers containing DSPS were prepared at π ≈ 30 mN/m, which is the highest surface pressure reliably achievable before reaching the collapse pressure (see isotherms in the Supporting Information). The solvent was allowed to evaporate for 20 min prior to any measurement. 2.2.2. Synthesis of Polyelectrolyte Brushes Grafted to Planar Silicon Substrates. Positively charged poly-METAC (PMETAC) polyelectrolyte brushes were grown from an initiator-modified silicon block by Atom Transfer Radical Polymerization (ATRP).27 The initiator 2-bromo-2-methyl-N-[3-(triethoxysilyl)-propyl]-propanamide (BTPAm) was synthesized according to the procedure reported in the literature.28 For the substrate cleaning and surface activation prior to any functionalization or reflectivity experiments, a common protocol was adopted, consisting of a treatment by piranha mixture (H2SO4:H2O2 1:1 v/v) (caution: highly corrosive and strong oxidizer, handle with care and do not store in closed containers) for 10 min and thorough rinsing with Milli-Q water. For the preparation of polyelectrolyte brushes, the substrate was dried in an oven at 70 °C for 30 min, then soaked in (1 × 10−3) mM BTPAm solution in toluene. The reaction was allowed to run for 24 h to allow for the formation of a tightly packed self-assembled monolayer (SAM) and was terminated by transferring the substrate into toluene and ethanol, followed by drying with a nitrogen stream. The thickness of the silane monolayer was approximately 8 Å as determined by ellipsometry measurements (Picometer light, Beaglehole, available at the PSCM, ILL) in ambient conditions (23 °C, 30% r.h.). The BTPAm-modified silicon substrate was used within 2 h of its functionalization for the preparation of polyelectrolyte brushes by the grafting-f rom approach. The reaction was run in a custom-made glass chamber which allows for constant nitrogen bubbling and sealing during polymerization.29 The polymerization protocol for PMETAC was adapted from ref 30. In brief, a polymerization mixture was prepared by dissolving 80 g of METAC (80%wt solution in water) in 64 g methanol and 16 g water under constant nitrogen bubbling, followed by addition of 2.40 g bipyridine, 0.61 g CuCl, and 0.04 g CuCl2. When a homogeneous mixture was formed, it was transferred in the reaction chamber, where the SAM-modified block were located under nitrogen atmosphere. The reaction was let run for 4 h and terminated by quenching in MilliQ water. The PMETAC-functionalized substrate was rinsed with methanol for 5 min under sonication and dried with a nitrogen stream. The thickness of the PMETAC brushes measured by ellipsometry in ambient condition was 160 ± 5 Å assuming a refractive index of n = 1.50.31 The sample was stored in a desiccator under inert atmosphere for 48 h prior to the experiments.

requires planar, smooth, and macroscopically large samples. Bringing two such macroscopic surfaces to a defined and homogeneous interaction distance is generally challenging. In pioneering studies on the structure of interacting soft interfaces, Kuhl and co-workers confined two end-grafted polystyrene brushes between solid substrates kept parallel at separations down to below 100 nm and characterized them by NR.16,17 In later studies on polymer brushes under confinement by Prescott and co-workers, one solid substrate was substituted with a flexible plastic membrane to facilitate alignment and the exertion of compression forces.18−20 Recently, Rodriguez Loureiro et al. carried out NR experiments on interacting nanometric lipid-anchored PEG brushes prepared by sequential Langmuir−Blodgett/Langmuir−Schaefer deposition onto planar substrates.21 The distance between the brush grafting surfaces was tuned by variation of the relative humidity, while their structure was deduced from the reflectivity data. However, none of the above approaches is free of limitations: while the use of two solid surfaces is intolerant to impurities and cannot easily reach nanometric surface separations, the use of a plastic membrane reduces the choice of surface functionalization and prevents the independent characterization by NR of each interface prior to the interaction. Control of the surface separation by adjustment of the humidity, on the other hand, prevents the establishment of large surface separations associated with conditions of weak interactions. Here we introduce an experimental setup which allows one to bring two arbitrary soft interfaces from noninteracting conditions at macroscopic separation to controlled interaction conditions. One of them is created by functionalization of a solid/liquid interface, the other one by functionalization of an interface between two immiscible liquids. Initially, each of them can be characterized by NR individually and subsequently the structural rearrangements associated with the establishment of mutual interactions can be determined. The feasibility of this approach is demonstrated for lipid-anchored uncharged hydrophilic polymer brushes interacting across water with silicon substrates both with and without functionalization with a charged polyelectrolyte brush. Different interaction scenarios are identified depending on the chemical composition of the involved interfaces. With the proposed method, we provide the scientific community with a new approach for the study of the distance-dependent structure of interacting soft interfaces. It can be applied to both technologically and biologically motivated questions. The latter can be tackled due to the availability of established methods for the preparation of realistic mimics of biological interfaces.22−26

2. EXPERIMENTAL SECTION 2.1. Materials. Silicon crystals (size (55 × 30 × 10) mm3 cut along the (111) crystalline plane) were purchased from Sil’Tronix (Archamps, France). The (111) orientation is preferred in our experiments due to the higher density of functional groups and higher homogeneity. The NR measurements revealed a surface roughness of around 4 Å. The monomer [2-(methacryloyloxy)ethyl]trimethylammonium chloride (METAC), copper(I)-chloride (CuCl), copper(II)-chloride (CuCl2), 2,2′-bipyridyl (≥99%), methanol (99.8%), chloroform, deuterated water (D2O), and dry dodecane (≥99.9%) were from Sigma-Aldrich (St. Quentin, France). All the products were used as received, without any further purification. Dodecane was used either as received, i.e., in a dehydrated state, or saturated with D2O. To prepare D2O-saturated dodecane, 50 mL of dodecane was transferred to a glass bottle and 1 mL D2O was added at least 1 month before use, to ensure the equilibration of the two phases. B

DOI: 10.1021/acs.langmuir.7b02971 Langmuir XXXX, XXX, XXX−XXX

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Langmuir 2.2.3. Realization of Controlled Interaction Conditions. Silicon blocks, either bare and freshly cleaned or functionalized with positively charged PMETAC polyelectrolyte brushes (see Section 2.2.2), were placed at the bottom of a custom-made quartz cell developed for reflectometry studies on liquid/liquid interfaces32 and covered with D2O. Lipid-anchored PEG brushes were deposited on top of the D2O phase by spreading defined amounts of lipid/PEG-lipid mixtures in organic solvents to reach the desired surface pressure for the given cell dimensions. After solvent evaporation, the bulk oil phase was added by carefully pouring dodecane onto the water surface using a syringe. At this point the two interfaces are still separated from each other by a water layer of several millimeters thickness. To bring them into contact, water was slowly released until a significant deformation of the oil/water meniscus was observed, indicating the inset of interfacial forces when the water layer thickness was no longer macroscopic (Figure 1B). To avoid the formation of water pockets trapped between

with bk being the coherent scattering length of an atomic nucleus of type k and Nik the number of such nuclei in the chemical component i occupying the volume vi. With that, the depth distribution of the molecular constituents in a sample can be reconstructed from the analysis of the R(qz) curves. In order to maximize the SLD contrast and thus the reflected intensity, deuterated water (D2O, ρD2O = 6.35 × 10−6 Å−2) was mostly used in this study. In addition, by changing the hydrogen-to-deuterium ratio for water and lipid alkyl chains, certain components were either highlighted or nullified.35 This was the case for the characterization of lipid/PEG-lipid monolayers at the air/water interface, where not only D2O, but also a H2O/D2O mixture known as air contrast matched water (ACMW, 92:8 H2O:D2O v/v) with ρACMW = 0, was used. 2.3.1. Air/Water Setup. Lipid-anchored PEG brushes in the form of lipid/PEG-lipid mixed monolayers were deposited either on D2O or on ACMW at defined lateral pressure as described in Section 2.2.1. The neutron beam reached the sample through air using two incident angles, θ1 = 0.62° and θ2 = 3.78°, and with wavelength range of 2 Å < λ < 20 Å. The f ull width at half-maximum resolution in qz, δqz/qz, was about 8%. All the measurements were carried out at room temperature. 2.3.2. Solid/Liquid Setup. The quartz cell introduced in Section 2.2.3 was used as the sample holder for all the measurements involving solid-grafted polyelectrolyte brushes, both in noninteracting conditions and when in contact with a functionalized oil/water interface. For these measurements D2O was always used as an aqueous phase, with the neutron beam reaching the solid/liquid interface from the bottom, passing through the denser and more transparent medium, i.e., the quartz wall of the sample cell and the silicon block. The measurements were carried out at room temperature using two incident angles, θ1 = 0.62° and θ2 = 2.70°, a wavelength range 2 Å < λ < 16 Å, and δqz/qz ≈ 8%. 2.3.3. Reflectivity Fitting Procedure. As detailed in the Results and Discussion section, the experimental reflectivity curves were interpreted in terms of structural models involving the interfacial distributions of all chemical components. To find the best-matching model parameters, sets of initial parameters were chosen to calculate the corresponding interfacial SLD profiles ρ(z) for each condition. The resulting ρ(z) profiles were then discretized into hundreds of thin slabs of 1 or 3 Å thickness and constant SLD. The corresponding reflectivity curves were then calculated using dynamical reflection theory and compared to the experimental data. To this end, the qzdependent reflection intensities were calculated via application of Fresnel’s reflection laws at each slab/slab interface using the iterative procedure of Parratt.36 To account for the finite experimental qz resolution, all the theoretical reflectivity curves calculated for the case of infinite resolution were convoluted with Gaussian functions representing the experimental resolution. In the last step, the parameters were varied until the best simultaneous agreement with all experimental reflectivity curves, characterized by the minimal chisquare deviation, χ2, was reached. Roughness values had a lower limit of 2 Å for interfaces involving solids (Si/SiO2 and SiO2/sil) and 5 Å for all ”soft” interfaces. Purely statistical errors corresponding to the 95% (two-sigma, 2σ) confidence interval were calculated for the most relevant parameters from the diagonal elements of the parameter covariance matrix. These are given in the Supporting Information. Note, however, that these estimates are valid only within the framework of a ”perfect model” and typically largely underestimate the real parameter uncertainties which also reflect uncertainties due to systematic errors. Throughout the main text of this manuscript, alternative error estimates are therefore provided. They roughly reflect the variation of the obtained parameters throughout the evolution and refinement of the employed model description, i.e., they reflect the robustness of the parameters with respect to the model, and we therefore consider them more meaningful.

Figure 1. Realization of controlled interaction conditions. (A) A planar solid substrate with functionalized surface is placed at the bottom of a quartz cell and covered with water. The oil/water interface is functionalized with an amphiphilic monolayer. Initially, the two interfaces are independent and macroscopically separated. (B) Water is released until the oil/water interface establishes contact with the solid surface. the two interfaces, the cell was titled at an angle of 3°. In this configuration the contact between the two interfaces was first established on one end of the block and then propagated homogeneously along the sample. When the contact was complete, the cell was tilted back horizontally and the reflectivity measurements were carried out. Due to the deformability of the interface the surface separation does not vary linearly with the water release. Instead, for a given water volume in the cell, the water layer thickness between the two surfaces is influenced by the interfacial forces. In the presence of an adhesive minimum the oil/water interface slightly deforms, such that the water layer thickness matches the optimal adhesion distance under bulk water condition, i.e., at zero osmotic pressure. However, in contrast to sample architectures involving an air/water interface, zeroosmotic-pressure-conditions can be maintained due to the slow water dynamics in oil. Moreover, as demonstrated in the Results and Discussion section, osmotic pressure can be used to reduce the water layer thickness below the equilibrium value under bulk water conditions. 2.3. Neutron Reflectometry Experiments. Specular neutron reflectometry (NR) was performed on the horizontal time-of-flight reflectometer FIGARO33 at the Institut Laue-Langevin (Grenoble, France), which is an instrument designed and optimized in particular for the investigation of structure and processes at fluid interfaces.34 In the experiments, the incident beam impinges onto the interface with incident angle θ. The reflectivity, i.e., the intensity ratio R between reflected and incident beams, is recorded as a function of the scattering vector component perpendicular to the interface, qz = (4π/λ)sin(θ), where λ is the neutron wavelength. The reflectivity R(qz) depends on the scattering length density (SLD) profile ρ(z) along the direction perpendicular to the interface, z. The SLD profile, in turn, originates from the interfacial distributions of all chemical components (see Results and Discussion section) having their characteristic SLDs

1 ρi = vi

∑

Nkibk

3. RESULTS AND DISCUSSION 3.1. Measurement Concept. The key ingredients of the experimental setup are the two individual surfaces of interest

(1) C

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Langmuir (see Figure 1A). One of them is based on a planar solid and can be realized via covalent functionalization,37 physisorption,38 or self-assembly.22 The other one is based on the interface between two immiscible liquids like oil and water and can be realized via adsorption of amphiphilic monolayers.39 The two surfaces are then brought into contact in a measurement cell as schematically illustrated in Figure 1B. The setup offers significant advantages for the structural investigation of interacting soft interfaces by reflectometry: (i) Each individual interface can be characterized prior to the interaction. (ii) Since one of the interfaces is flexible, a homogeneous interaction distance is always realized and tolerant to local perturbations, for instance, due to micron-sized dust particles. This is in contrast to approaches involving two planar solids, where creation of a defined interaction distance is generally challenging.16,17 (iii) As demonstrated further below, the oil layer prevents undesired water evaporation and thus allows fully hydrated interaction conditions to be maintained over long periods. The interaction distance can be purposefully reduced via exertion of osmotic pressure. In the following sections, the measurement concept is illustrated for a system of two interacting polymer brushes: an uncharged, lipid-anchored PEG brush immobilized at a water/ dodecane interface (Figure 2A) and a positively charged PMETAC polyelectrolyte brush terminally grafted to a silicon substrate (Figure 2E). This system was chosen because it is asymmetrical, contains a variety of different chemical components, spontaneously forms a weakly adhered state, and exhibits significant structural response to changes in the surface separation. First, the structural characterization by NR of the two single surfaces under noninteracting conditions will be presented, followed by the structural characterization of two such surfaces under various interaction conditions. A global model is used in order to interpret all reflectivity curves in a consistent manner, notably conserving the amounts of the involved chemical components. 3.2. Structural Characterization of a Lipid-Anchored PEG Brush. A lipid-anchored PEG brush immobilized at the air/water interface at π ≈ 45 mN/m was prepared as described in the Experimental Section. The chain-deuterated phospholipid d-DSPC was used as matrix lipid anchoring PEG-lipid at a mole fraction of f = 0.1. Each PEG chain comprises NPEG = 114 monomers of volume vEG = 69 Å3 and linear dimension (segment length) of aEG = 4.1 Å.21,40 The brush was characterized by NR using two water subphases, D2O and ACMW. The use of deuterated alkyl chains enhances the scattering contrast between the lipid chains and their surroundings. Figure 2D shows the experimental reflectivity curves obtained with both water contrasts. The solid lines represent the simulated reflectivity curves based on the bestmatching parameters in the common model described below. A pronounced Kiessig oscillation with Δqz ≈ 0.04 Å−1 is visible in the D2O contrast, indicating the presence of a thick layer (≈ 160 Å) with considerably different SLD to that of pure D2O. The shape of the reflectivity curve obtained with ACMW is governed by the layer of deuterated hydrocarbon chains, which gives excellent signal when sandwiched between two media with null SLD. To analyze the reflectivity data, a theoretical model schematically illustrated in Figure 2B was invoked. It is based on the volume fraction profiles perpendicular to the interface, ϕi(z), of all chemical components, i.e., air (i = ”air”), deuterated lipid hydrocarbon chains (i = ”dhc”), lipid headgroups (i =

Figure 2. (A and E) Schematic representation of the two soft interfaces investigated individually, a lipid-anchored PEG brush at the air/water interface (A) and an end-grafted PMETAC polyelectrolyte brush at the silicon/water interface (E). (B and F) Schematic illustration of the theoretical models used to interpret the experimental reflectivity curves from the lipid-anchored PEG brush (B) and the solid-grafted PMETAC brush (F), respectively, shown in Panels D and H. The model is based on the volume fraction profiles ϕ(z) of all chemical components. (C and G) Best-matching profiles obtained in the reflectivity fits and according to the solid lines superimposed to the reflectivity data in Panels D and H. Labels: deuterated lipid hydrocarbon chains ”dhc”, lipid head groups ”hg”, PEG brush ”PEG”, water ”wat”, silicon substrate ”Si”, silicon oxide ”SiO2”, silane ”sil”, PMETAC brush ”PME”.

”hg”), PEG (i = ”PEG”), and water (i = ”wat”). The corresponding SLD profile ρ(z) follows as ρ(z) = ϕair(z)ρair + ϕdhc(z)ρdhc + ϕhg (z)ρhg + ϕPEG(z)ρPEG + ϕwat(z)ρwat

(2)

where z denotes the distance measured along the normal to the planar sample surface and z = 0 is chosen to be the interface between the lipid headgroup layer and the aqueous medium accommodating the PEG brush. As in earlier studies,21,40,41 D

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For the thicknesses of the alkyl chain and headgroup layers we obtain ddhc = 15 ± 1 Å and dhg = 10 ± 1 Å, respectively, in good agreement with earlier reports.44,45 For the adjustable SLD of the alkyl chain layer, ρdhc = (7.5 ± 0.5) × 10−6 Å−2 is obtained, which is rationalized considering a mixture of 90% deuterated hydrocarbons and 10% hydrogenous hydrocarbons and the condensation effects of the liquid condensed phase, consistent with published values of molecular volumes.46 The obtained PEG volume per unit area, given by DPEG = 14 ± 1 Å, corresponds to an area per lipid Alip = f NPEGvEG/DPEG ≈ 56 Å, significantly larger than the area per lipid in a pure DSPC monolayer at the same surface pressure (ADSPC = 47 Å).47 This lip observation is consistent with earlier reports21 and seems to reflect the significant lateral repulsion exerted by the overlapping polymers. The obtained brush grafting density σ is encoded in DPEG as σ = DPEG/(NPEGvEG) = f/Alip ≈ 1.8 × 10−3 Å−2. According to the Alexander−deGennes model for neutral polymer brushes,48,49 the brush regime applies in the case of σ/ σOT ≫ 1, where σOT = RF−2 is the overlap threshold and RF = aN3/5 PEG ≈ 70 Å is the Flory radius. Here, with σ/σOT ≈ 8, the brush regime applies. Regarding the volume fraction profile of the PEG brush we obtain Λ = 105 ± 5 Å and n = 2.0 ± 0.2 for the decay length and the stretching/compression exponent, respectively. As seen in Figure 2C, the profile is reminiscent of the parabolic shape predicted by self-consistent-field (SCF) theory,50 but also features a tail at the brush periphery as seen in computer simulations.51 A quantitative comparison of the brush profile with SCF theory is provided in the Supporting Information. It yields the estimate pτ ≈ 0.9 for the product of the number p of monomers in a persistent segment and the reduced temperature τ. This value is significantly larger than the one estimated earlier for longer but more sparsely grafted PEG brushes.40 3.3. Structural Characterization of a Surface-Grafted PMETAC Brush. The PMETAC brush end-grafted to a planar silicon substrate (Figure 2E) was characterized by NR in the solid/liquid setup described in the Experimental Section. Figure 2H shows the experimental reflectivity curve obtained in D2O. The critical edge of total reflection at qz ≈ 0.015 Å−1 is characteristic of the reflection from a Si/D2O interface. The presence of a pronounced Kiessig oscillation with Δqz ≈ 0.03 Å−1 confirms the presence of a layer of about 200 Å with considerably different SLD to that of D2O. The solid line represents the simulated reflectivity curve based on the best-matching parameters, according to the model schematically illustrated in Figure 2F. As explained in the previous section, the SLD profile follows from the volume fraction profiles ρi(z) of all chemical components. For this sample we have silicon (i = ”Si”), silicon oxide (i = ”SiO2”), silan (BTPAm, i = ”sil”), PMETAC (i = ”PME”), and water (i = ”wat”)

ideal mixing of all components is assumed, requiring that the sum of all volume fractions in the model amounts to one at each z-position

∑ ϕi(z) ≡ 1

(3)

The air medium is modeled as semi-infinite continuum with null SLD (ρair = 0). The lipid hydrocarbon chain and headgroup layers are represented as homogeneous slabs with adjustable thicknesses ddhc and dhg and characteristic SLDs ρdhc and ρhg for hydrocarbon chains and head groups, respectively. The latter is fixed to the literature value ρhg = 1.75 × 10−6 Å−2,42 while ρdhc is an adjustable parameter allowing for small variations in the chain packing density. The water content in the lipid headgroup layer is described with an adjustable parameter, ϕhg wat, defining the relative water fraction in this layer. In contrast, the water fraction in the hydrophobic hydrocarbon layer is assumed to be zero. To account for interfacial roughness, the slab profiles are modulated by error functions with adjustable roughness parameters δi/j. The SLD of PEG is fixed to the literature value, ρPEG = 0.6 × 10−6 Å−2.43 The volume fraction of the PEG brush anchored to the lipid monolayer is modeled as a stretched/compressed exponential function n

ϕPEG(z) = Ihg/wat(z) ·ϕ0 e−| z / Λ|

(4)

where Ihg/wat(z) accounts for the topographic roughness of the anchoring surface in the form of an error function characterized by the roughness parameter δhg/wat. In eq 4, the parameters ϕ0, Λ, and n denote the maximal volume fraction of the monomer distribution, its decay length, and the stretching/compression exponent, respectively. This functional form is chosen because by the change of n a wide spectrum of shapes can be described with a minimum number of independent parameters. To quantify the amount per area of each component, with exception for the bulk media, an equivalent thickness Di is defined +∞

Di =

∫−∞

ϕi(z) dz

(5)

which introduces additional constraints based on the known stoichiometry of the various chemical components. More specifically, it is possible to constrain the ratio between DPEG and Dhg from the mole fraction f of PEG-lipids in the monolayer, the polymerization degree NPEG, and the molecular volumes vEG and vhg of ethylene glycol monomers and lipid head groups, respectively21 DPEG = D hg

fNPEGvEG v hg

(6)

In other words, for a given set of the adjustable parameters Λ and n in eq 4 the parameter ϕ0 is not free, but it follows from Dhg according to eq 6. Finally, the water volume fraction profile ϕwat(z) follows from all other distributions according to eq 3. Figure 2C shows the volume fraction profiles according to the best-matching model parameters, corresponding to the solid lines in Figure 2D. As implied by the monolayer composition, the alkyl chains are narrowly distributed at the air/water interface, followed by the head groups immersed in the aqueous phase and the extended and highly hydrated PEG chains anchored to the lipid head groups. The profiles also constitute the basis for the schematic illustration in Figure 2A.

ρ(z) = ϕSi(z)ρSi + ϕSiO (z)ρSiO + ϕsil(z)ρsil 2

2

+ ϕPME(z)ρPME + ϕwat(z)ρwat

(7)

The position z = 0 is defined as the interface between the silane and the aqueous medium accommodating the hydrated PMETAC brush. The silicon substrate is modeled as semiinfinite continuum with constant SLD (ρSi = 2.07 × 10−6 Å−2). The SiO2 and silane layers are represented as homogeneous slabs with adjustable thicknesses dSiO2 and dsil, respectively, and interfacial roughness δSi/SiO2 and δSiO2/sil. The SLD of SiO2 is E

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Langmuir kept at the literature value ρSiO2 = 3.47 × 10−6 Å −2. However, in line with earlier reports,41 we account for oxide hydration effects by allowing for a finite water content in the SiO2 layer, 2 characterized by an adjustable parameter ϕSiO wat . The SLD of the silane layer, ρsil, in lack of a reliable estimate is a free parameter, and its water content is assumed to be negligible. For the SLD of PMETAC we use ρPME = 0.8 × 10−6 Å−2, as follows from a recent report on the monomer volume vME = 262 Å3.52 To describe the volume fraction profile of PMETAC brushes terminally grafted to the silane interface, several functional forms were tested. Neither a slab representation nor a single stretched/compressed exponential (as for the PEG brush in the previous section) were sufficient to adequately reproduce the reflectivity curve. However, the experimental data could be well described with a two-component description, where ϕPME(z) = in ϕinPME(z) + ϕout PME(z). The inner region ϕPME(z) is represented by a homogeneous slab characterized by the parameters dinPME, PME δin/out PME , and ϕ0 , which denote thickness, roughness, and the maximal volume fraction in the slab, respectively. The outer part of the profile, ϕout PME(z), describes a decaying region represented by a stretched/compressed exponential function (see eq 4) with parameters for the decay length and the stretching exponent of the distribution, respectively. Figure 2G shows the volume fraction profiles according to the best-matching model parameters, corresponding to the solid lines in Figure 2H. The profiles also constitute the basis for the schematic illustration in Figure 2E. The inner region of the polyelectrolyte brush, with thickness dinPME = 190 ± 10 Å, comprises most of the total polymer amount. The outer region exhibits a characteristic decay length of Λ = 200 ± 15 Å and a stretching/compression exponent of n = 0.9 ± 0.2, where the latter is much lower than observed for the uncharged PEG brush in the previous section. The overall amount of PMETAC as extracted from the volume fraction profile according to eq 5 is DPME = 126 ± 5 Å, which is in reasonable agreement with the thickness delli = 160 ± 5 Å measured using ellipsometry at ambient conditions (23 °C, 30% r.h.), given that the deviation between DPME and delli can be attributed to a minor yet considerable water content in the brush at ambient conditions. The two-region profile of the PMETAC brush is characteristic of polyelectrolyte brushes created using the graf ting from approach.53,54 As pointed out by Titmuss and co-workers,55 the presence of a dense inner region can be explained by the hydrophobic interactions between backbone and silane, as well as by a high density of shorter chains. The slowly decaying periphery of the profile, corresponding to the low stretching/ compression exponent appears to reflect considerable polydispersity also among the longer chains. Generally, polyelectrolyte brushes are more stretched than uncharged brushes, due to electrostatic interactions, which can also be understood in terms of the osmotic pressure of the counterions.56 In contrast to the monodisperse PEG brush characterized in the previous section, the parameters of the PMETAC brush in terms of grafting density and average monomer number are not known a priori, but can only be roughly estimated from the volume fraction profile. Given the molecular structure of METAC (Figure S1 in the Supporting Information), the monomer was modeled as a cuboid with dimensions [aME(width) × aME(depth) × 3aME (length of the side group)]. With the molecular volume per METAC monomer vME = 262 Å3 = (aME × aME × 3aME), the resulting segment length is aME = 4.4 Å. The extension H of the PMETAC brush

is roughly approximated as the thickness of the inner region plus the decay length of the outer region, H ≈ dinPME + Λ ≈ 400 Å. According to Zhulina et al.,56 the extension of a fully charged polyelectrolyte brush is approximately the average contour length of the polymer chains, H ≈ aN, where N is the monomer number. Accordingly we roughly obtain NPME ≈ H/aME ≈ 90 for the average monomer number. Finally, the average grafting density is roughly estimated from the relation σ = DPME/ (vMENPME), which yields σPME ≈ 5 × 10−3 Å−2. 3.4. Structural Characterization of Two Interacting Interfaces. The structural characterization by NR of the two individual surfaces, the lipid-anchored PEG brush and the solidgrafted polyelectrolyte brush, provided precise information on the configurations of the unperturbed brushes and on the overall interfacial amounts of several chemical components, notably of PMETAC. In the following we consider two such surfaces under interaction conditions. To this end, the PMETAC brush characterized in the previous section was brought into contact with a lipid-anchored PEG brush freshly prepared at the D2O/dodecane interface as described in the Experimental Section. The PEG brush was similar to the one characterized in the previous section, but in order to promote spontaneous adhesion, the zwitterionic matrix lipid DSPC was substituted with DSPS, which has the same alkyl chains but a negatively charged headgroup. It therefore carries the opposite charge of the PMETAC brush, so that considerable electrostatic attraction is generated. While the difference in the monolayer structure limits slightly the following comparison of the behavior of two interacting interfaces with the available data discussed above (Section 3.2), it may be noted that the two lipid species exhibit comparable area per lipid and the behavior of PEG brushes is rather robust with respect to minor variations of the grafting density.40,41 With respect to the PEG brush, it is expected that the structural properties in the brush regime, like chain extension and conformation, will be dominated by the solvation properties, intra- and intermolecular interactions, which barely change by variation of lipid matrix in this specific case. As discussed above, to prevent evaporation of water from the nanometric hydration layer, D2O-saturated dodecane oil was used to cover the lipid monolayer. According to earlier reports the presence of oil has no dramatic influence on the monolayer configuration.57 The interaction condition was realized by releasing D2O from the quartz cell (see Experimental Section). When the oil/ water interface reached the silicon surface, a region with significantly different optical reflectance appeared starting from the more elevated side of the tilted block, suggesting the formation of an adhered state with a water layer thickness far below the wavelength of visible light.58 NR was measured when the adhered state was homogeneous over the whole surface. The upper curve in Figure 3A shows the experimental neutron reflectivity data measured with the two interfaces in adhesion contact. Total reflectivity does not occur, a clear indication that the aqueous layer is no longer macroscopically thick. Instead, a series of pronounced periodic Kiessig fringes separated by Δqz ≈ 0.016 Å−1 indicates an overall layer thickness of ≈ 400 Å between the silicon block and the oil. The solid line superimposed to the experimental data represents the simulated reflectivity curve based on the best-matching model parameters according to the model schematically illustrated in Figure 3B. Analogous to the previous cases, the description of the system is based on the volume fraction profiles of all chemical components F

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water fraction ϕhg wat and in contact with the oil phase with SLD ρoil = −0.4 × 10−6 Å−2. The two surfaces of the headgroup slab are smeared out by error functions accounting for interfacial roughness, characterized by a single adjustable roughness parameter δwat/oil. Due to the low interfacial tension of the lipid-functionalized oil/water interface, the thermal fluctuations are much larger than those of the air/water interface or the roughness of the solid surface. Therefore, the smearing of the profiles of water, PEG, head groups, and oil around this interface is substantial. In order to retain full consistency with the structural properties of the single PMETAC brush prior to the interaction (Section 3.3), the PMETAC amount DPME as well as all parameters describing Si, SiO2, and silane layers were global parameters in the common model. For the description of the PMETAC profile under all interaction conditions investigated here, however, it was sufficient to use a single stretched/compressed exponential function, which can be attributed to the compression of the dilute brush periphery onto the inner region upon interaction with in opposing surface. Dhg and DPEG are again coupled parameters (eq 6). Figure 3C shows the volume fraction profiles according to the best-matching model parameters corresponding to the solid lines in Figure 3A. A layer of highly hydrated interacting brushes is confined between the solid substrate on one side and the dodecane phase on the other side, with a grafting surface separation d = 350 ± 10 Å. Within this gap, the two brushes overlap significantly, in line with the formation of an adhered state upon electrostatic attraction. The PEG amount obtained according to eq 6, DPEG = 25 ± 5 Å, is significantly higher than that of the DSPC-anchored PEG brush in Section 3.2. This can be partially understood from the lateral packing density of PSlipids, which was shown to be significantly higher than that of PC-lipids.59 Additional potential explanations include the nonnegligible experimental uncertainty in the PEG-lipid mole fraction f and monolayer corrugations induced by the electrostatic interactions with the PMETAC brush, resulting in an effectively higher packing projected to the surface normal. We refrain, however, from further interpreting this observation in view of significant uncertainties in the determination of DPEG. Namely, PEG and PMETAC in their hydrogenous forms have similar SLDs, so the robustness of the obtained amounts of each individual component is limited. In future experiments such limitation can be readily circumvented by contrast variation via solvent exchange, or by selective deuteration, as already demonstrated17,21 even when only one water contrast was used. In the present configuration, NR is mainly sensitive to the water distribution between the two brush grafting surfaces. The individual PEG and PMETAC profiles shown in Figure 3C and D therefore are not unique. Instead, only the combined ”PMETAC+PEG” profile can be considered robust. Some aspects of the polymer distributions are nevertheless reliable: the dilute peripheral region of the PMETAC brush is compressed onto the inner part, resulting in a maximal PMETAC volume fraction of ≈0.50, much higher than the corresponding value for the noninteracting case (≈0.33). Regarding the degree of mutual PMETAC and PEG brush interpenetration, an earlier work21 demonstrated using selective deuteration that PEG brushes strongly interpenetrate at this grafting density. Here, the PMETAC brush is, if at all, partially in contact with the lipid monolayer. In fact, only weak contact is expected theoretically, although the direct interaction of the polycationic polymers with the negatively charged monolayer can be assumed to be favorable with respect to counterion

Figure 3. (A) Experimental reflectivity data of the two soft interfaces, the lipid-anchored PEG brush and the solid-grafted PMETAC brush, in contact at full hydration (top) and under dehydrated conditions (bottom). For clarity the bottom curve is vertically shifted by multiplication with a factor of 0.05. The solid lines represent the simulated reflectivities according to the best-matching parameters in the common model. (B) Schematic illustrations of the theoretical model used to interpret the reflectivity data. The model is based on the volume fraction profiles ϕ(z) of all chemical components. (C and D) Best-matching profiles at full hydration (C) and under dehydrated conditions (D) obtained in the reflectivity fits and according to the solid lines in Panel A. Labels: silicon substrate ”Si”, silicon oxide ”SiO2”, silane ”sil”, PMETAC brush ”PME”, PEG brush ”PEG”, water ”wat”, lipid head groups ”hg”, dodecane ”oil”. The individual volume fraction profiles of PMETAC and PEG are not unique because of their similar SLDs. The measurements are mostly sensitive to the combined ”PME+PEG” profiles. (E and F) Schematic illustration of the sample configuration at full hydration (E) and under dehydrated conditions (F).

ρ(z) = ϕSi(z)ρSi + ϕSiO2(z)ρSiO2 + ϕsil(z)ρsil + ϕPME(z)ρPME + ϕwat(z)ρwat + ϕPEG(z)ρPEG + ϕhg (z)ρhg + ϕoil(z)ρoil

(8)

The hydrogenous lipid alkyl chains were not considered explicitly in the model given the lack of contrast with the oil phase.44 As for the measurements on the PMETAC brush alone, the position z = 0 is defined as the interface between the silane layer and the aqueous medium accommodating the hydrated polymers. The surface separation d is defined as the distance between the two brush grafting surfaces. Accordingly, the profile of the PEG brush is described with a mirrored version of eq 4, shifted along the z-axis by an increment d. The hydrated headgroup layer anchoring the PEG brush is described as a slab of idealized thickness dhg = 10 Å, containing G

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Langmuir interactions.60 By considering the charge neutralization between lipid headgroups and METAC monomers, the monolayer charge is compensated when Nbind ≈ 7 monomers are in contact with it, according to the relation Nbind ≈ σlip/σPME = DPEG/( f NPEGvegσPME). This elucidates why no strong compression is required for an electrostatically favorable configuration. In fact, the maximal water fraction between the two surfaces, located roughly in the overlap region of the two brushes, is similar to that in the inner region of the uncompressed PMETAC brush (≈0.65, see Section 3.3). Another point of note is the high roughness of the oil/water interface (δwat/oil = 20 ± 5 Å). As pointed out before, it is much higher than that of the air/water and solid liquid interfaces discussed in the previous sections. The high roughness is representative of a low interfacial tension γ, given that δwat/oil ∝ γ−1/2,61 and it further suggests a weak confinement between the oil/water interface on one side and the solid surface on the other side, allowing for considerable interfacial fluctuations. Altogether, the experimental results suggest that the interaction is dominated by the interfacial forces rather than by external dehydration forces, consistent with the complete D2O saturation of the dodecane oil. The structure of the system is dominated by the balance of two opposite forces: a weak, longrange electrostatic attraction due to the opposite surface charge of the opposing layers, and polymer-induced repulsion that prevents the brush collapse. The contribution of van der Waals attraction between the bulk media (silicon and dodecane interacting across water) was estimated from the Lifshitz theory for the “two-body” potential62 and found to be of minor relevance. In the present study, water was used without the addition of salt. Electrostatic interactions thus take place in a counterion-only environment:63 most of the counterions are entropically released into the bulk water reservoir, while in the interfacial water layer only overall charge neutrality is maintained. As a result, no ion excess is available for the screening of electrostatic interactions. The situation therefore corresponds to the limit of unscreened, strong electrostatic attraction until mutual charge compensation between the surface bound charges (polycationic PMETAC and negatively charged lipids) is achieved. The effect of added salt is an important aspect that should be studied in the future. In light of the importance of electrostatic attraction, significantly weaker adhesion or a ”no adhesion” scenario would be expected when noncharged lipids are used instead of DSPS. In the next step, osmotic stress was used to reduce the water amount within the layer, i.e., the separation between the two brush grafting surfaces.10,64 To this end, the D2O-saturated dodecane was largely replaced with dehydrated dodecane. The corresponding shift in the water chemical potential μ of the oil phase, Δμ = kBTln([D2O]dehyd/[D2O]sat), leads to the exertion of a dehydrating osmotic pressure of magnitude Π=−

k T [D2 O]dehyd Δμ = − B ln [D2 O]sat vw vw

The reflectivity curve obtained after the exchange with dehydrated dodecane and after 1 h equilibration is shown in Figure 3A. The curve did not evolve significantly during the measurement, which demonstrates that equilibrium was essentially reached at this time point. The observed fast equilibration (in less than 1 h) can be attributed to the fact that most of the initially water-saturated oil was replaced with dry oil. The larger Δqz of the Kiessig oscillations directly indicates a decrease of total layer thickness associated with the dehydration. The fit of the experimental data under the constraint of conserved DPME and DPEG yields the volume fraction profiles shown in Figure 3D. The surface separation is reduced from d ≈ 350 Å as deduced from the fit in the weakly adhered state (where ”weak” refers to the fact that the interfaces remain highly hydrated) to d = 230 ± 10 Å in the dehydrated state and the interfacial region is strongly dehydrated. The associated decrease in the equivalent water layer thickness Dw from 203 ± 5 Å to 82 ± 5 Å upon the application of the osmotic stress is substantial. While the ordered structure and the overall shapes of the volume fraction profiles are qualitatively preserved, both polyelectrolyte brush and the PEG chains are compressed and the maximal water fraction is reduced to ∼0.4, much lower that in the uncompressed brushes. With δwat/oil = 25 ± 5 Å the roughness of the oil/water interface remains similar to the corresponding value in the fully hydrated state. In this particular experiment, [D2O]dehyd was not known, due to mixing with the remaining saturated dodecane. In the following, we will therefore roughly estimate the dehydrating osmotic pressure from the structural response of the PMETAC brush, which occupies most of the hydrated region. The extension of the compressed brush, approximated from the zposition of its half-maximal volume fraction, is HPME compr ≈ 200 Å. The compression of a fully charged polyelectrolyte brush to an extension H requires a dehydrating pressure of Π(H ) ≃ kBT

σN H

(10)

where N is the monomer number.56,65 For H = HPME compr, N = NPME, and σ = σPME we obtain Π ≈ 100 bar. Although this is merely a rough estimate, the order of magnitude confirms that considerable equivalent pressures can be generated by purposefully dehydrating the oil phase. Over a long equilibration time, the oil is expected to saturate again with D2O, but the time scale for this equilibration process depends on several factors, including the mobility of water in oil. This notion suggests that the saturation kinetics can be controlled by the choice of the oil. It further suggests that the water saturation of the oil can be used as a parameter to vary the surface separation on long time scales, which allows one to monitor the structural response to the interfacial forces by reflectometry in situ. To this end, one should be able to achieve a slow and gradual reduction of the hydration level from full hydration to dehydrated conditions by adding dry oil dropwise. When an excess of D2O is pumped back in the chamber, the oil/water interface is withdrawn from the surface. The key features of the reflectivity curve of the noninteracting PMETAC brush are recovered (Figure S3 in the Supporting Information), while the deformation of the curve at low qz can be attributed to beam attenuation by residual oil traces on the wall of the sample cell. This result indicates that the interaction of the studied interfaces and the associated structural changes are reversible and the original structure can be restored.

(9)

where vw is the partial molecular volume of water, kB is the Boltzmann constant, T the temperature, [D2O]dehyd the water concentration in the dehydrated dodecane, and [D2O]sat is the water concentration in water-saturated dodecane. A significant advantage of the present measurement setup is the control of the hydration level of oil for longer spatiotemporal scales in comparison to air, because of the much slower water dynamics in the liquid medium. H

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Langmuir 3.5. Other Interaction Scenarios. The system described in the previous section exhibits a highly hydrated adhered state in the absence of dehydrating pressures as a result of its particular interfacial force balance. In this section, other interaction scenarios are presented, which were generated by varying the molecular composition at the interfaces. 3.5.1. Dry Adhesion. A lipid-anchored PEG brush at the dodecane/water interface was allowed to interact with a bare Si/SiO2 surface, i.e., without a polyelectrolyte brush. Like the case of the PEG brush characterized at the air/water interface, the lipid matrix consisted of zwitterionic DSPC mixed with PEG-lipid at mole fraction f = 0.1. The lateral density of the monolayer was matched to achieve a surface pressure π ≈ 45 mN/m, as described in the Experimental Section, and dehydrated dodecane was used. Once the two surfaces were placed in contact, the formation of an adhered state was clearly visible from a noticeable change of the optical reflectance. Figure 4A shows the experimental neutron reflectivity curves of

consistently set to the volume-weighted average of PEG and headgroup SLDs, ρorg = 0.95 × 10−6 Å−2. Figure 4B shows the volume fraction profiles corresponding to the best matching model parameters. The hydrated organic layer has an extension of dorg ≈ 100 Å and is highly infiltrated with dodecane. Interestingly, the water fraction in the layer is very low, ≈ 5%, corresponding to ϕorg wat ≈ 13% in the oil-free volume. We recall that these measurements are very sensitive to the water fraction because of the high SLD of D2O. The low hydration level can be attributed on the one hand partially to the dehydrating effect of the dry dodecane, and on the other hand to the formation of a strongly adhesive state between the SiO2 surface and the amphiphilic monolayer hosting the PEGchains. In fact, the adhesion of PC lipid head groups to SiO2 and metal oxides is documented to be strong. Even in the presence of PEG lipids, the PC head groups adsorb to silica42 and alumina,66 so that PEG is not acting as a hydrated cushion between the solid substrate and the lipid layer, but it is displaced and intermixed with the lipids. These reports are in line with the present observation of a poorly hydrated and intermixed layer of organic material adsorbed to the solid surface. Furthermore, the significant intermixing of PEG with the hydrophobic dodecane is consistent with the pronounced surface activity of PEG at the hydrophobic air/water interface.67 We remark that this dry adhesion scenario is robust with respect to the exact value of Dorg within a plausible range. Van der Waals attraction between the bulk media may be a considerable contribution to the interfacial force balance in this scenario. 3.5.2. No Adhesion. As described in the previous paragraph, strong adhesion occurs between a bare Si/SiO2 surface and a PEG-brush anchored to a DSPC monolayer at the oil/water interface. The deep adhesive minimum can be attributed to favorable interactions between SiO2 and the PC head groups of DSPC. Such an adhesive minimum is suppressed when DSPC is replaced with negatively charged DPPS. One of the reasons for the repulsion between SiO2 and the negatively charged PS headgroup is that silica is also negatively charged after the cleaning procedure by piranha-treatment.68 The suppression of an adhesive minimum for this surface formulation manifests already during sample preparation, when bringing the surfaces into contact by releasing water. In contrast to the scenarios discussed above, no change in the optical reflectance is observed upon surface contact, suggesting that the thickness of the water layer remains larger than or comparable to the wavelength of visible light. Figure 5 shows experimental neutron reflectivity data in D2O of the Si/SiO2 surface before (open squares) and after (open circles) the approach of the DPPS-anchored PEG brush. The critical edge of total reflection at the qz position characteristic of the Si/D2O (qz ≈ 0.015 Å−1) interface is there also after the approach, which is an indication that the neutron beam is essentially still probing an Si/SiO2/D2O interface. The simulated reflectivity of an ideal Si/SiO2/D2O interface is indicated with a red dashed line. Nevertheless, there is an important change in the reflectivity upon surface approach: the increase of reflectivity above the critical edge, most strongly in the range 0.016 Å−1 < qz < 0.03 Å−1, which could be explained only by the contribution of the neutrons reflected from the lipid-anchored PEG brush at the D2O/dodecane interface. This observation suggests that this interface is indeed a microscopic distance from the solid and aligned with it. The absence of Kiessig fringes prevents the precise thickness determination, but a lower estimate can be given: for the used qz-resolution as

Figure 4. (A) Reflectivity data of an Si/SiO2/D2O interface before (open square) and after (open circle) adhesion of a lipid-anchored PEG brush at the oil/water interface. The dash-dotted line represents the simulated reflectivity for an ideal Si/SiO2/dodecane interface. (B) Best-matching volume fraction profiles when the lipid-anchored PEG brush is adhered to the solid substrate. The phospholipid/PEG-lipid mixed monolayer is described as a homogeneous organic layer adhered onto the silicon substrate with very low water content and infiltrated with dodecane.

the Si/SiO2 surface in D2O before (open squares) and after adhesion (open circles) of the lipid-anchored PEG brush. Prior to adhesion, the data have the typical shape of a silicon/D2O interface, merely featuring the critical edge of total reflection at qz ≈ 0.015 Å−1. Upon adhesion, the reflectivity drops dramatically and becomes closer to that of an ideal silicon/ dodecane interface, which is modeled in Figure 4A as a red, dashed line, but it also exhibits Kiessig oscillations originating from the adsorbed monolayer. The solid lines in Figure 4A represent the simulated reflectivity curves based on the bestmatching model parameters for the Si/SiO2 surface before and after monolayer adhesion. The common parameters concern the thickness and roughness of the SiO2 slab, with dSiO2 ≈ 16 Å and δSi/SiO2 ≈ 5.0 Å. The reflectivity data after adhesion could be reproduced with a simple slab representation of the hydrated organic material (i.e., DSPC and PEG-lipid) adjacent to the SiO2 surface and infiltrated with dodecane. The slab of organic material has an adjustable thickness dorg, water content ϕorg wat, and outer roughness δorg/oil. For the amount of organic material, Dorg = Dhg + DPEG ≈ 20 Å, we assume DPEG and Dhg to be equal to the respective values obtained for the lipid-anchored PEG brush characterized at the air/water interface, which had the same composition. The SLD of the organic material is I

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Before and after contact, the two interfaces can be structurally characterized independently to identify the structural changes associated with interaction. The feasibility of this approach using NR was demonstrated for lipid-anchored PEG brushes interacting with a bare Si/SiO2 surface and with a Si/SiO2 surface displaying a polyelectrolyte brush. Depending on the chemical nature of the interfaces, a wide spectrum of interaction scenarios is observed, ranging from nearly dry adhesion to no adhesion. For a PEG brush interacting with a solid-grafted polyelectrolyte brush, weak adhesion occurs, with the two hydrated brushes interpenetrating. When osmotic stress is applied, the layers get further compressed as a consequence of a significant dehydration. The volume fraction profiles of the chemical components, as deduced self-consistently from the reflectivity data for both hydration levels, provide insight into the distancedependent structure of these soft and complex interacting interfaces and allow the identification of the structure of the individual components as a function of surface separation or interaction pressure, respectively. Such detailed structural information on the interacting surfaces can be obtained whenever the water layer thickness is no larger than several hundreds of nanometers, depending on the qz-resolution of the instrument setup. This requirement is fulfilled for all systems exhibiting spontaneous adhesion, like in most scenarios relevant for membrane adhesion studies. For systems exhibiting purely repulsive interactions, additional measures can be taken in order to bring the surfaces into a small-enough separation. The use of opposite charges, as demonstrated here, is one approach, but not the only one. The impact of the proposed method is its wide applicability to investigations of the structure and the interactions of soft interfaces in general, ranging from biologically to technologically relevant configurations. Potential applications include studies on the interaction of biological membranes in terms of nonspecific and specific adhesion, as well as molecular exchange. When it comes to wet-technological studies, the present method may help elucidate the lateral densities and distributions of particles or solutes confined between two interfaces as well as the physical mechanisms of foam stability.

Figure 5. Reflectivity data of a Si/SiO2/D2O interface before (open square) and after (open circle) the approach of a PEG brush anchored to a negatively charged lipid monolayer. The reflectivity data after withdrawal of the PEG brush are indicated with filled circles. The solid line represents the simulated reflectivity of an ideal Si/SiO2/D2O interface.

determined by the instrument settings used, such fringes are resolved and visible only when the water layer thickness is below ≈3500 Å. Conversely, their absence indicates that the water layer is likely to be thicker than this threshold value. As seen in Figure 5, the original reflectivity is recovered when the surfaces are again brought apart to macroscopic separations by pumping in an excess amount of D2O (filled circles). Interestingly, the no adhesion scenario occurs for this formulation irrespective of the water saturation level of the oil. This behavior can be rationalized in terms of the water mobility: water molecules in the thin layer can exchange with two different environments, namely, (i) with the oil phase, in which the water chemical potential is determined by the water saturation (see eq 9), and (ii) with the surrounding bulk water phase, which has the reference chemical potential corresponding to zero osmotic pressure. The water chemical potential in the thin layer depends on the water exchange rate with the two environments. While the exchange across the monolayer with the oil phase is independent of the water layer thickness, the one with the bulk water phase strongly increases with the water layer thickness, because the channel for lateral diffusion becomes wider. For large-enough surface separations, the chemical potential is thus governed by the bulk water phase, so that no osmotic dehydration is exerted by the oil phase. In contrast, exchange with the oil phase is dominant for thin water layers, as was the case for the adhesive scenarios described in the previous sections, where the osmotic dehydration by the oil phase was achieved. In future studies when salt is added to the aqueous phase, a local increase in the salt concentration upon water extraction through dehydrated oil may have to be considered.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.7b02971.

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4. SUMMARY AND CONCLUSIONS We have introduced a novel experimental setup for the study of soft interfaces under controlled interaction conditions. It involves a functionalized solid surface interacting across an aqueous film with a functionalized oil/water interface. The water layer thickness is primarily governed by the interfacial forces, but it can be varied through the exertion of dehydrating osmotic pressures under typical conditions. Planar geometry and macroscopic size of the contact region enable the use of reflectivity techniques to resolve the molecular-scale structure of the interacting soft interfaces depending on their separation.

Molecular structures, pressure−area isotherms, comparison with SCF theory, purely statistical parameter errors, and additional neutron reflectometry data (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Yuri Gerelli: 0000-0001-5655-8298 Richard A. Campbell: 0000-0002-6296-314X Emanuel Schneck: 0000-0001-9769-2194 Notes

The authors declare no competing financial interest. J

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ACKNOWLEDGMENTS The authors thank for financial support the Max Planck Society (MPG) and by the German Federal Ministry of Research and Education (BMBF) within the Röntgen-Ångström cluster (Grant 05K16ECA). The Institut Laue-Langevin is acknowledged for providing beamtime (LTP-9-7),69 the Partnership for Soft Condensed Matter (PSCM) at ILL for the use of chemistry laboratories, equipment and instruments, and Giovanna Fragneto for the scientific support within the Soft Matter Science group. We acknowledge Franck Cecillion for help with the experimental setup and Ernesto Scoppola, Bruno Demé, and Helmuth Möhwald for fruitful discussions. E.S. acknowledges support from an Emmy-Noether grant (SCHN 1396/1) of the German Research Foundation (DFG).

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