Illuminating the nanostructure of diffuse interfaces: Recent advances and future directions in reflectometry techniques.

Diffuse soft matter interfaces take many forms, from end-tethered polymer brushes or adsorbed surfactants to self-assembled layers of lipids. These interfaces play crucial roles across a multitude of fields, including materials science, biophysics, and nanotechnology. Understanding the nanostructure and properties of these interfaces is fundamental for optimising their performance and designing novel functional materials. In recent years, reflectometry techniques, in particular neutron reflectometry, have emerged as powerful tools for elucidating the intricate nanostructure of soft matter interfaces with remarkable precision and depth. This review provides an overview of selected recent developments in reflectometry and their applications for illuminating the nanostructure of diffuse interfaces. We explore various principles and methods of neutron and X-ray reflectometry, as well as ellipsometry, and discuss advances in their experimental setups and data analysis approaches. Improvements to experimental neutron reflectometry methods have enabled greater time resolution in kinetic measurements and elucidation of diffuse structure under shear or confinement, while innovation in analysis protocols has significantly reduced data processing times, facilitated co-refinement of reflectometry data from multiple instruments and provided greater-than-ever confidence in proposed structural models. Furthermore, we highlight some significant research findings enabled by these techniques, revealing the organisation, dynamics, and interfacial phenomena at the nanoscale. We also discuss future directions and potential advancements in reflectometry techniques. By shedding light on the nanostructure of diffuse interfaces, reflectometry techniques enable the rational design and tailoring of interfaces with enhanced properties and functionalities.

Solvent-Modulated Specific Ion Effects: Poly(N-isopropylacrylamide) Brushes in Nonaqueous Electrolytes.

Pertinent to cryopreservation as well as energy storage and batteries, nonaqueous electrolytes and their mixtures with water were investigated. In particular, specific ion-induced effects on the modulation of a poly(N-isopropylacrylamide) (PNIPAM) brush were investigated in various dimethyl sulfoxide (DMSO)-water solvent mixtures. Spectroscopic ellipsometry and neutron reflectometry were employed to probe changes in brush swelling and structure, respectively. In water-rich solvents (i.e., pure water and 6 mol % DMSO), PNIPAM undergoes a swollen to collapsed thermotransition with increasing temperature, whereby a forward Hofmeister series was noted; K+ and Li+ electrolytes composed of SCN- and I- salted-in (stabilized) PNIPAM chains, and electrolytes of Cl- and Br- salted-out (destabilized) the polymer. The cation was seen to play a lesser role than that of the anion, merely modulating the magnitude of the anion effect. In 70 mol % DMSO, a collapsed to swollen thermotransition was noted for PNIPAM. Here, concentration-dependent specific ion effects were observed; a forward series was observed in 0.2 mol % electrolytes, whereas increasing the electrolyte concentration to 0.9 mol % led to a series reversal. While no thermotransition was observed in pure DMSO, a solvent-induced specific ion series reversal was noted; SCN- destabilized the brush and Cl- stabilized the brush. Both series reversals are attributed to the delicate balance of interactions between the solvent, solute (ion), and substrate (brush). Namely, the stability of the solvent clusters was hypothesized to drive polymer solvation.

Underscreening in concentrated electrolytes: re-entrant swelling in polyelectrolyte brushes.

Hypersaline environments are ubiquitous in nature and are found in myriad technological processes. Recent empirical studies have revealed a significant discrepancy between predicted and observed screening lengths at high salt concentrations, a phenomenon referred to as underscreening. Herein we investigate underscreening using a cationic polyelectrolyte brush as an exemplar. Poly(2-(methacryloyloxy)ethyl)trimethylammonium (PMETAC) brushes were synthesised and their internal structural changes and swelling response was monitored with neutron reflectometry and spectroscopic ellipsometry. Both techniques revealed a monotonic brush collapse as the concentration of symmetric monovalent electrolyte increased. However, a non-monotonic change in brush thickness was observed in all multivalent electrolytes at higher concentrations, known as re-entrant swelling; indicative of underscreening. For all electrolytes, numerical self-consistent field theory predictions align with experimental studies in the low-to-moderate salt concentration regions. Analysis suggests that the classical theory of electrolytes is insufficient to describe the screening lengths observed at high salt concentrations and that the re-entrant polyelectrolyte brush swelling seen herein is consistent with the so-called regular underscreening phenomenon.

Nanostructure Explains the Behavior of Slippery Covalently Attached Liquid Surfaces.

Slippery covalently-attached liquid surfaces (SCALS) with low contact angle hysteresis (CAH, <5°) and nanoscale thickness display impressive anti-adhesive properties, similar to lubricant-infused surfaces. Their efficacy is generally attributed to the liquid-like mobility of the constituent tethered chains. However, the precise physico-chemical properties that facilitate this mobility are unknown, hindering rational design. This work quantifies the chain length, grafting density, and microviscosity of a range of polydimethylsiloxane (PDMS) SCALS, elucidating the nanostructure responsible for their properties. Three prominent methods are used to produce SCALS, with characterization carried out via single-molecule force measurements, neutron reflectometry, and fluorescence correlation spectroscopy. CO2 snow-jet cleaning was also shown to reduce the CAH of SCALS via a modification of their grafting density. SCALS behavior can be predicted by reduced grafting density, Σ, with the lowest water CAH achieved at Σ≈2. This study provides the first direct examination of SCALS grafting density, chain length, and microviscosity and supports the hypothesis that SCALS properties stem from a balance of layer uniformity and mobility.

Exploring the water capture efficiency of covalently attached liquid-like surfaces.

The capture of moisture from the atmosphere through condensation has the potential to provide a sustainable source of water. Here, we investigate the condensation of humid air at low subcooling condition (11 °C), similar to conditions for natural dew capture, and explore how water contact angle and contact angle hysteresis affect the rates of water capture. We compare water collection on three families of surfaces: (i) hydrophilic (polyethylene oxide, MPEO) and hydrophobic (polydimethylsiloxane, PDMS) molecularly thin coatings grafted on smooth silicon wafers, which produce slippery covalently attached liquid surfaces (SCALSs), with low contact angle hysteresis (CAH = 6°); (ii) the same coatings grafted on rougher glass, with high CAH (20°-25°); (iii) hydrophilic polymer surfaces [poly(N-vinylpyrrolidone), PNVP] with high CAH (30°). Upon exposure to water, the MPEO SCALS swell, which likely further increases their droplet shedding ability. MPEO and PDMS coatings collect similar volume of water (around 5 l m-2 day-1), both when they are SCALS and non-slippery. Both MPEO and PDMS layers collect about 20% more water than PNVP surfaces. We present a basic model showing that, under low heat flux conditions, on all MPEO and PDMS layers, the droplets are so small (600-2000 µm) that there is no/low heat conduction resistance across the droplets, irrespective of the exact value of contact angle and CAH. As the time to first droplet departure is much faster on MPEO SCALS (28 min) than on PDMS SCALS (90 min), slippery hydrophilic surfaces are preferable in dew collection applications where the collection time frame is limited.

Spatz: the time-of-flight neutron reflectometer with vertical sample geometry at the OPAL research reactor.

The Spatz neutron beam instrument is the second time-of-flight neutron reflectometer to be installed at the OPAL research reactor. The instrument was formerly the V18 BioRef reflectometer at the BER-II reactor in Berlin and was transferred to Australia in 2016. Subsequently the instrument was re-installed in the neutron guide hall of the OPAL reactor at the end position of the CG2B cold-neutron guide and recommissioned. The instrument performance has not been compromised by the move, with reflectivity achieved down to 10-7 and good counting statistics within a reasonable time frame using a wavelength range of 2-20 Å. Several different samples at the solid-air interface and the solid-liquid interface have been measured to demonstrate the instrument's capabilities.

Advice on describing Bayesian analysis of neutron and X-ray reflectometry.

As a result of the availability of modern software and hardware, Bayesian analysis is becoming more popular in neutron and X-ray reflectometry analysis. The understandability and replicability of these analyses may be harmed by inconsistencies in how the probability distributions central to Bayesian methods are represented in the literature. Herein advice is provided on how to report the results of Bayesian analysis as applied to neutron and X-ray reflectometry. This includes the clear reporting of initial starting conditions, the prior probabilities, the results of any analysis and the posterior probabilities that are the Bayesian equivalent of the error bar, to enable replicability and improve understanding. It is believed that this advice, grounded in the authors' experience working in the field, will enable greater analytical reproducibility in the work of the reflectometry community, and improve the quality and usability of results.

From Hofmeister to hydrotrope: Effect of anion hydrocarbon chain length on a polymer brush.

HYPOTHESIS: Specific ion effects govern myriad biological phenomena, including protein-ligand interactions and enzyme activity. Despite recent advances, detailed understanding of the role of ion hydrophobicity in specific ion effects, and the intersection with hydrotropic effects, remains elusive. Short chain fatty acid sodium salts are simple amphiphiles which play an integral role in our gastrointestinal health. We hypothesise that increasing a fatty acid's hydrophobicity will manifest stronger salting-out behaviour. EXPERIMENTS: Here we study the effect of these amphiphiles on an exemplar thermoresponsive polymer brush system, conserving the carboxylate anion identity while varying anion hydrophobicity via the carbon chain length. Ellipsometry and quartz crystal microbalance with dissipation monitoring were used to characterise the thermoresponse and viscoelasticity of the brush, respectively, whilst neutron reflectometry was used to reveal the internal structure of the brush. Diffusion-ordered nuclear magnetic resonance spectroscopy and computational investigations provide insight into polymer-ion interactions. FINDINGS: Surface sensitive techniques unveiled a non-monotonic trend in salting-out ability with increasing anion hydrophobicity, revealing the bundle-like morphology of the ion-collapsed system. An intersection between ion-specific and hydrotropic effects was observed both experimentally and computationally; trending from good anti-hydrotrope towards hydrotropic behaviour with increasing anion hydrophobicity, accompanying a change in hydrophobic hydration.

Effect of surfactants on the thermoresponse of PNIPAM investigated in the brush geometry.

HYPOTHESIS: Anionic surfactants have been reported to interact with poly(N-isopropyl acrylamide) (PNIPAM), suppressing its thermoresponse. Scattering and NMR studies of the anionic sodium dodecylsulfate (SDS) system propose that the PNIPAM-surfactant interaction is purely hydrophobic. However, prior phenomenological investigations of a range of surfactant identities (anionic, cationic, nonionic) show that only anionic surfactants affect the thermoresponse and conformation of PNIPAM, implying that the hydrophilic head-group also contributes. Crucially, the phenomenological experiments do not measure the affinity of the tested surfactants to the polymer, only their effect on its behaviour. EXPERIMENTS: We study the adsorption of six surfactants within a planar PNIPAM brush system, elucidating the polymer conformation, thermoresponse, and surfactant adsorption kinetics using ellipsometry, neutron reflectometry (NR), optical reflectometry and the quartz crystal microbalance technique. NR is used to measure the distribution of surfactants within the brush. FINDINGS: We find that only anionic surfactants modify the structure and thermoresponse of PNIPAM, with the greater affinity of anionic surfactants for PNIPAM (relative to cationic and nonionic surfactants) being the primary reason for this behaviour. These results show that the surfactant head-group has a more critical role in mediating PNIPAM-surfactant interaction than previously reported. Taking inspiration from prior molecular dynamics work on the PEO-surfactant system, we propose an interaction mechanism for PNIPAM and SDS that reconciles evidence for hydrophobic interaction with the observed head-group-dependent affinity.

The Assembly Mechanism and Mesoscale Architecture of Protein-Polysaccharide Complexes Formed at the Solid-liquid Interface.

Protein-polysaccharide composite materials have generated much interest due to their potential use in medical science and biotechnology. A comprehensive understanding of the assembly mechanism and the mesoscale architecture is needed for fabricating protein-polysaccharide composite materials with desired properties. In this study, complex assemblies were built on silica surfaces through a layer-by-layer (LbL) approach using bovine beta-lactoglobulin variant A (ßLgA) and pectin as model protein and polysaccharide, respectively. We demonstrated the combined use of quartz crystal microbalance with dissipation monitoring (QCM-D) and neutron reflectometry (NR) for elucidating the assembly mechanism as well as the internal architecture of the protein-polysaccharide complexes formed at the solid-liquid interface. Our results show that ßLgA and pectin interacted with each other and formed a cohesive matrix structure at the interface consisting of intertwined pectin chains that were cross-linked by ßLgA-rich domains. Although the complexes were fabricated in an LbL fashion, the complexes appeared to be relatively homogeneous with ßLgA and pectin molecules spatially distributed within the matrix structure. Our results also demonstrate that the density of ßLgA-pectin complex assemblies increased with both the overall and local charge density of pectin molecules. Therefore, the physical properties of the protein-polysaccharide matrix structure, including density and level of hydration, can be tuned by using polysaccharides with varying charge patterns, thus promoting the development of composite materials with desired properties.

Using refnx to Model Neutron Reflectometry Data from Phospholipid Bilayers.

Neutron reflectometry has emerged as a powerful method for studying the structure of thin films in contact with solution at sub-molecular spatial resolution (Penfold and Thomas, J Phys Condens Matter 2:1369-1412, 1990). This type of experiment is undertaken at large international central facilities and experience in data analysis and interpretation is not always available "locally". Here, we describe the application of the refnx software suite (Nelson and Prescott, J Appl Crystallogr 52:193-200, 2019) to the analysis of a single phospholipid bilayer deposited at a silicon/buffer interface. The data is modeled such that the fitted parameters are readily interpretable by researchers working with lipid bilayers.

Structure and Hydration of Asymmetric Polyelectrolyte Multilayers as Studied by Neutron Reflectometry: Connecting Multilayer Structure to Superior Membrane Performance.

Porous membranes coated with so-called asymmetric polyelectrolyte multilayers (PEMs) have recently been shown to outperform commercial membranes for micropollutant removal. They consist of open support layers of poly(styrene sulfonate) (PSS)/poly(allylamine) (PAH) capped by denser and more selective layers of either PAH/poly(acrylic acid) (PAA) or PAH/Nafion. Unfortunately, the structure of these asymmetric PEMs, and thus their superior membrane performance, is poorly understood. In this work, neutron reflectometry (NR) is employed to elucidate the multilayered structure and hydration of these asymmetric PEMs. NR reveals that the multilayers are indeed asymmetric in structure, with distinct bottom and top multilayers when air-dried and when solvated. The low hydration of the top [PAH/Nafion] multilayer, together with the low water permeance of comparable [PAH/Nafion]-capped PEM membranes, demonstrate that it is a reduction in hydration that makes these separation layers denser and more selective. In contrast, the [PAH/PAA] capping multilayers are more hydrated than the support [PSS/PAH] layers, signifying that, here, densification of the separation layer occurs through a decrease in the mesh size (or effective pore size) of the top layer due to the higher charge density of the PAH/PAA couple compared to the PSS/PAH couple. The [PAH/PAA] and [PAH/Nafion] separation layers are extremely thin (∼4.5 and ∼7 nm, respectively), confirming that these asymmetric PEM membranes have some of the thinnest separation layers ever achieved.

Author Correction: SciPy 1.0: fundamental algorithms for scientific computing in Python.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

SciPy 1.0: fundamental algorithms for scientific computing in Python.

SciPy is an open-source scientific computing library for the Python programming language. Since its initial release in 2001, SciPy has become a de facto standard for leveraging scientific algorithms in Python, with over 600 unique code contributors, thousands of dependent packages, over 100,000 dependent repositories and millions of downloads per year. In this work, we provide an overview of the capabilities and development practices of SciPy 1.0 and highlight some recent technical developments.

refnx: neutron and X-ray reflectometry analysis in Python.

refnx is a model-based neutron and X-ray reflectometry data analysis package written in Python. It is cross platform and has been tested on Linux, macOS and Windows. Its graphical user interface is browser based, through a Jupyter notebook. Model construction is modular, being composed from a series of components that each describe a subset of the interface, parameterized in terms of physically relevant parameters (volume fraction of a polymer, lipid area per molecule etc.). The model and data are used to create an objective, which is used to calculate the residuals, log-likelihood and log-prior probabilities of the system. Objectives are combined to perform co-refinement of multiple data sets and mixed-area models. Prior knowledge of parameter values is encoded as probability distribution functions or bounds on all parameters in the system. Additional prior probability terms can be defined for sets of components, over and above those available from the parameters alone. Algebraic parameter constraints are available. The software offers a choice of fitting approaches, including least-squares (global and gradient-based optimizers) and a Bayesian approach using a Markov-chain Monte Carlo algorithm to investigate the posterior distribution of the model parameters. The Bayesian approach is useful for examining parameter covariances, model selection and variability in the resulting scattering length density profiles. The package is designed to facilitate reproducible research; its use in Jupyter notebooks, and subsequent distribution of those notebooks as supporting information, permits straightforward reproduction of analyses.

Biomineralization of Calcium Phosphate and Calcium Carbonate within Iridescent Chitosan/Iota-Carrageenan Multilayered Films.

This work systematically explores the biomineralization of calcium phosphate (CaP) and carbonate (CaCO3) within chitosan/iota-carrageenan multilayer films. Multilayer films of chitosan and iota-carrageenan (up to 128-coupled layers) were prepared on glass substrates by a layer-by-layer dip-coating technique. Cryo-scanning electron microscopy revealed dense interfaces between the chitosan and iota-carrageenan layers with thicknesses in the range 250 and 350 nm in the hydrated state, accounting for the iridescent nature of multilayer films when wet. Immersion of the multilayered films in simulated body fluid or simulated seawater at 25 °C resulted in the mineralization of CaP and CaCO3, respectively, at the interfaces between the biopolymer layers and modified the iridescence of the films. Lamellar scattering features in small-angle neutron scattering measurements of the mineralized films provided evidence of the localized mineralization. Further evidence of this was found through the lack of change in the dynamic and static correlation lengths of the polymer networks within the bulk phase of the chitosan and iota-carrageenan layers. CaP mineralization occurred to a greater extent than CaCO3 mineralization within the films, evidenced by the higher lamellar density and greater rigidity of the CaP-mineralized films. Results provide valuable new insights into CaP and CaCO3 biomineralization in biopolymer networks.

Surface engineering of poly(methylmethacrylate): Effects on fluorescence immunoassay.

The authors present surface engineering modifications through chemistry of poly(methylmethacrylate) (PMMA) that have dramatic effects on the result of surface-bound fluorescence immunoassays, both for specific and nonspecific signals. The authors deduce the most important effect to be clustering of antibodies on the surface leading to significant self-quenching. Secondary effects are attributable to the formation of sparse multilayers of antibody. The authors compare PMMA as an antibody support surface with ultraviolet-ozone oxidized PMMA and also to substrates that were, after the oxidation, surface modified by a four-unit poly(ethyleneglycol) carboxylic acid (PEG4), a branched tricarboxylic acid, and a series of carboxylic acid-terminated dendrimers, from generation 1.5 to 5.5. Fluorescence immunoassay and neutron reflectometry were used to compare the apparent antibody surface loading, antigen binding and nonspecific binding on these various surfaces using anti-human IgG as a model antibody, chemically coupled to the surface by amide formation. Simple physical adsorption of the antibody on PMMA resulted in a thick antibody multilayer with small antigen binding capacity. On the carboxylated surfaces, with chemical coupling, a simple monolayer was formed. The authors deduce that antibody clustering was driven by conformational inflexibility and high carboxylate density. The PEG4-modified surface was the most conformationally flexible. The dendrimer-modified interfaces showed a collapse and densification. In fluorescence immunoassay, the optimal combination of high specific and low nonspecific fluorescence signal was found for the G3.5 dendrimer.

Using Neutron Reflectometry to Characterize Antimicrobial Protein Surface Coatings.

Understanding the interaction of adsorbed or covalently immobilized proteins with solid substrates at the molecular level guides the successful design of functionalized surfaces used in biomedical applications. In this report, neutron reflectometry (NR) was used to characterize the structure of surface-attached antimicrobial protein films, with antimicrobial activity assessed using an adaption of the Japanese Industrial Standard Test JIS Z 2801. NR allowed parameters influencing bioactivity to be measured at nanometer resolution and for conclusions about structural characteristics relating to bioactivity to be drawn. Hydramacin-1 (HM-1) and lysozyme were covalently attached to poly(methyl methacrylate) (PMMA) and 3-aminopropyltriethoxysilane (APTES) films in the presence and absence of a four-unit poly(ethylene glycol) PEG-based spacer and measured using NR, followed by antimicrobial assays. APTES-PEG-protein films were structurally unique, with a layer of 80% water directly beneath the protein layer, and were the only films that displayed antimicrobial activity against Escherichia coli and Bacillus subtilis. The hydration content of these films combined with the subtle difference in the PEG layer thickness of APTES versus PMMA films played a role in defining antimicrobial activity of the prepared surface coatings.

Oxidative Damage to Biomimetic Membrane Systems: In Situ Fe(II)/Ascorbate Initiated Oxidation and Incorporation of Synthetic Oxidized Phospholipids.

Damage to cellular membranes from oxidative stress has been implicated in aging related diseases. We report the effects of oxidative damage on the structure and properties of biomimetic phospholipid membrane systems. Two oxidation methods were used, in situ oxidation initiated using Fe(II) and ascorbate, and the incorporation of a synthetic "oxidized" phospholipid, PoxnoPC, into biomimetic membranes. The biomimetic systems employed included multibilayer stacks, tethered bilayers, and phospholipid monolayers studied using a combination of reflectometry, attenuated total reflection infrared spectroscopy, electrochemical impedance spectroscopy, and neutron diffraction. We show that oxidation with Fe(II) and ascorbate caused an increase in the order of the membrane, attributed to cross-linking of the phospholipids, and a change in the electrical permeability of the membrane, but no significant impact on the thickness or completeness of the membrane. Incorporation of PoxnoPC, on the other hand, had a larger impact on the structure of the membrane. Inversion of the aldehyde-terminated truncated sn-2 chain of PoxnoPC into the head group region was observed, along with a slight decrease in the thickness and order of the membrane.

Morphological, chemical and kinetic characterisation of zein protein-induced biomimetic calcium phosphate films.

This study examines zein protein-induced growth of calcium phosphate (CaP) thin films at air-liquid interfaces. The results demonstrate that zein protein films in contact with simulated body fluids (SBF) promote the growth of hemispherical CaP particles of 150 nm radius, which combine to form a film. The CaP films formed on zein were less continuous than those obtained using a hexadecanoic acid monolayer self-assembled at the air-liquid interface. In situ ellipsometry measurements were used to follow the nucleation and growth of CaP in these systems. The kinetic parameters extracted from the ellipsometry data were dependent on the organic layer and also the SBF concentration. The mineralisation process was slower and final CaP film thicknesses smaller when zein films were used. XPS analyses revealed the presence of zein protein only on the air-facing side of the CaP films, confirming that the zein protein is involved in nucleating the unusual hemispherical CaP morphology. The zein protein-induced CaP thin films showed iridescence with a distinct range of colours as a function of CaP film thickness. Templated CaP mineralisation with zein protein is a simple and chemically facile method for synthesising CaP films with novel morphologies and controllable thicknesses.