Synthetic and biopolymeric microgels: Review of similarities and difference in behaviour in bulk phases and at interfaces.

This review discusses the current knowledge of interfacial and bulk interactions of biopolymeric microgels in relation to the well-established properties of synthetic microgels for applications as viscosity modifiers and Pickering stabilisers. We present a timeline showing the key milestones in designing microgels and their bulk/ interfacial performance. Poly(N-isopropylacrylamide) (pNIPAM) microgels have remained as the protagonist in the synthetic microgel domain whilst proteins or polysaccharides have been primarily used to fabricate biopolymeric microgels. Bulk properties of microgel dispersions are dominated by the volume fraction (ϕ) of the microgel particles, but ϕ is difficult to pinpoint, as addressed by many theoretical models. By evaluating recent experimental studies over the last five years, we find an increasing focus on the analysis of microgel elasticity as a key parameter in modulating their packing at the interfaces, within the provinces of both synthetic and biopolymeric systems. Production methods and physiochemical factors shown to influence microgel swelling in the aqueous phase can have a significant impact on their bulk as well as interfacial performance. Compared to synthetic microgels, biopolymer microgels show a greater tendency for polydispersity and aggregation and do not appear to have a core-corona structure. Comprehensive studies of biopolymeric microgels are still lacking, for example, to accurately determine their inter- and intra- particle interactions, whilst a wider variety of techniques need to be applied in order to allow comparisons to real systems of practical usage.

Identification of membrane engineering targets for increased butanol tolerance in Clostridium saccharoperbutylacetonicum.

There is a growing interest in the use of microbial cell factories to produce butanol, an industrial solvent and platform chemical. Biobutanol can also be used as a biofuel and represents a cleaner and more sustainable alternative to the use of conventional fossil fuels. Solventogenic Clostridia are the most popular microorganisms used due to the native expression of butanol synthesis pathways. A major drawback to the wide scale implementation and development of these technologies is the toxicity of butanol. Various membrane properties and related functions are perturbed by the interaction of butanol with the cell membrane, causing lower yields and higher purification costs. This is ultimately why the technology remains underemployed. This study aimed to develop a deeper understanding of butanol toxicity at the membrane to determine future targets for membrane engineering. Changes to the lipidome in Clostridium saccharoperbutylacetonicum N1-4 (HMT) throughout butanol fermentation were investigated with thin layer chromatography and mass spectrometry. By the end of fermentation, levels of phosphatidylglycerol lipids had increased significantly, suggesting an important role of these lipid species in tolerance to butanol. Using membrane models and in vitro assays to investigate characteristics such as permeability, fluidity, and swelling, it was found that altering the composition of membrane models can convey tolerance to butanol, and that modulating membrane fluidity appears to be a key factor. Data presented here will ultimately help to inform rational strain engineering efforts to produce more robust strains capable of producing higher butanol titres.

Transforming sustainable plant proteins into high performance lubricating microgels.

With the resource-intensive meat industry accounting for over 50% of food-linked emissions, plant protein consumption is an inevitable need of the hour. Despite its significance, the key barrier to adoption of plant proteins is their astringent off-sensation, typically associated with high friction and consequently poor lubrication performance. Herein, we demonstrate that by transforming plant proteins into physically cross-linked microgels, it is possible to improve their lubricity remarkably, dependent on their volume fractions, as evidenced by combining tribology using biomimetic tongue-like surface with atomic force microscopy, dynamic light scattering, rheology and adsorption measurements. Experimental findings which are fully supported by numerical modelling reveal that these non-lipidic microgels not only decrease boundary friction by an order of magnitude as compared to native protein but also replicate the lubrication performance of a 20:80 oil/water emulsion. These plant protein microgels offer a much-needed platform to design the next-generation of healthy, palatable and sustainable foods.

PDMS as a Substrate for Lipid Bilayers.

PDMS (polydimethylsiloxane) is a cheap, optically clear polymer that is elastic and can be easily and quickly fabricated into a wide array of microscale and nanoscale architectures, making it a versatile substrate for biophysical experiments on cell membranes. It is easy to imagine many new experiments will be devised that require a bilayer to be placed upon a substrate that is flexible or easily cast into a desired geometry, such as in lab-on-a-chip, organ-on-chip, and microfluidic applications, or for building accurate membrane models that replicate the surface structure and elasticity of the cytoskeleton. However, PDMS has its limitations, and the extent to which the behavior of membranes is affected on PDMS has not been fully explored. We use AFM and fluorescence optical microscopy to investigate the use of PDMS as a substrate for the formation and study of supported lipid bilayers (SLBs). Lipid bilayers form on plasma-treated PDMS and show free diffusion and normal phase transitions, confirming its suitability as a model bilayer substrate. However, lipid-phase separation on PDMS is severely restricted due to the pinning of domains to surface roughness, resulting in the cessation of lateral hydrodynamic flow. We show the high-resolution porous structure of PDMS and the extreme smoothing effect of oxygen plasma treatment used to hydrophilize the surface, but this is not flat enough to allow domain formation. We also observe bilayer degradation over hour timescales, which correlates with the known hydrophobic recovery of PDMS, and establish a critical water contact angle of 30°, above which bilayers degrade or not form at all. Care must be taken as incomplete surface oxidation and hydrophobic recovery result in optically invisible membrane disruption, which will also be transparent to fluorescence microscopy and lipid diffusion measurements in the early stages.

Frictional behaviour of plant proteins in soft contacts: unveiling nanoscale mechanisms.

Despite the significance of nanotribology in the design of functional biomaterials, little is known about nanoscale friction in the presence of protein-coated soft contact surfaces. Here, we report a detailed investigation of frictional behaviour of sustainable plant proteins at the nanoscale for the first time, using deformable bio-relevant surfaces that achieve biologically relevant contact pressures. A combination of atomic force microscopy, quartz crystal microbalance with dissipation monitoring, and friction force microscopy with soft polydimethylsiloxane (PDMS, 150 kPa) surfaces was employed to elucidate the frictional properties of model plant proteins, i.e. lupine, pea, and potato proteins at the nanoscale while systematically varying the pH and ionic strength. Interactions of these plant proteins with purified mucins were also probed. We provide the much-needed direct experimental evidence that the main factor dictating the frictional properties of plant proteins is their affinity towards the surface, followed by the degree of protein film hydration. Proteins with high surface affinity, such as pea and potato protein, have better lubricating performance than lupine at the nanoscale. Other minor factors that drive lubrication are surface interactions between sliding bodies, especially at low load, whilst jamming of the contact area caused by larger protein aggregates increases friction. Novel findings reveal that interactions between plant proteins and mucins lead to superior lubricating properties, by combining high surface affinity from the plant proteins and high hydration by mucins. We anticipate that fundamental understanding gained from this work will set the stage for the design of a plethora of sustainable biomaterials and food with optimum nanolubrication performance.

Self-Quenching Behavior of a Fluorescent Probe Incorporated within Lipid Membranes Explored Using Electrophoresis and Fluorescence Lifetime Imaging Microscopy.

Fluorescent probes are useful in biophysics research to assess the spatial distribution, mobility, and interactions of biomolecules. However, fluorophores can undergo "self-quenching" of their fluorescence intensity at high concentrations. A greater understanding of concentration-quenching effects is important for avoiding artifacts in fluorescence images and relevant to energy transfer processes in photosynthesis. Here, we show that an electrophoresis technique can be used to control the migration of charged fluorophores associated with supported lipid bilayers (SLBs) and that quenching effects can be quantified with fluorescence lifetime imaging microscopy (FLIM). Confined SLBs containing controlled quantities of lipid-linked Texas Red (TR) fluorophores were generated within 100 × 100 µm corral regions on glass substrates. Application of an electric field in-plane with the lipid bilayer induced the migration of negatively charged TR-lipid molecules toward the positive electrode and created a lateral concentration gradient across each corral. The self-quenching of TR was directly observed in FLIM images as a correlation of high concentrations of fluorophores to reductions in their fluorescence lifetime. By varying the initial concentration of TR fluorophores incorporated into the SLBs from 0.3% to 0.8% (mol/mol), the maximum concentration of fluorophores reached during electrophoresis could be modulated from 2% up to 7% (mol/mol), leading to the reduction of fluorescence lifetime down to 30% and quenching of the fluorescence intensity down to 10% of their original levels. As part of this work, we demonstrated a method for converting fluorescence intensity profiles into molecular concentration profiles by correcting for quenching effects. The calculated concentration profiles have a good fit to an exponential growth function, suggesting that TR-lipids can diffuse freely even at high concentrations. Overall, these findings prove that electrophoresis is effective at producing microscale concentration gradients of a molecule-of-interest and that FLIM is an excellent approach to interrogate dynamic changes to molecular interactions via their photophysical state.

Fibrin protofibril packing and clot stability are enhanced by extended knob-hole interactions and catch-slip bonds.

Fibrin polymerization involves thrombin-mediated exposure of knobs on one monomer that bind to holes available on another, leading to the formation of fibers. In silico evidence has suggested that the classical A:a knob-hole interaction is enhanced by surrounding residues not directly involved in the binding pocket of hole a, via noncovalent interactions with knob A. We assessed the importance of extended knob-hole interactions by performing biochemical, biophysical, and in silico modeling studies on recombinant human fibrinogen variants with mutations at residues responsible for the extended interactions. Three single fibrinogen variants, γD297N, γE323Q, and γK356Q, and a triple variant γDEK (γD297N/γE323Q/γK356Q) were produced in a CHO (Chinese Hamster Ovary) cell expression system. Longitudinal protofibril growth probed by atomic force microscopy was disrupted for γD297N and enhanced for the γK356Q mutation. Initial polymerization rates were reduced for all variants in turbidimetric studies. Laser scanning confocal microscopy showed that γDEK and γE323Q produced denser clots, whereas γD297N and γK356Q were similar to wild type. Scanning electron microscopy and light scattering studies showed that fiber thickness and protofibril packing of the fibers were reduced for all variants. Clot viscoelastic analysis showed that only γDEK was more readily deformable. In silico modeling suggested that most variants displayed only slip-bond dissociation kinetics compared with biphasic catch-slip kinetics characteristics of wild type. These data provide new evidence for the role of extended interactions in supporting the classical knob-hole bonds involving catch-slip behavior in fibrin formation, clot structure, and clot mechanics.

Why fibrin biomechanical properties matter for hemostasis and thrombosis.

Polymeric fibrin displays unique structural and biomechanical properties that contribute to its essential role of generating blood clots that stem bleeds. The aim of this review is to discuss how the fibrin clot is formed, how protofibrils make up individual fibrin fibers, what the relationship is between the molecular structure and fibrin biomechanical properties, and how fibrin biomechanical properties relate to the risk of thromboembolic disease. Fibrin polymerization is driven by different types of bonds, including knob-hole interactions displaying catch-slip characteristics, and covalent crosslinking of fibrin polypeptides by activated factor XIII. Key biophysical properties of fibrin polymer are its visco-elasticity, extensibility and resistance to rupture. The internal packing of protofibrils within fibers changes fibrin biomechanical behavior. There are several methods to analyze fibrin biomechanical properties at different scales, including AFM force spectroscopy, magnetic or optical tweezers and rheometry, amongst others. Clinically, fibrin biomechanical characteristics are key for the prevention of thromboembolic disorders such as pulmonary embolism. Future studies are needed to address unanswered questions regarding internal molecular structure of the fibrin polymer, the structural and molecular basis of its remarkable mechanical properties and the relationship of fibrin biomechanical characteristics with thromboembolism in patients with deep vein thrombosis and ischemic stroke.

Elimination of fibrin γ-chain cross-linking by FXIIIa increases pulmonary embolism arising from murine inferior vena cava thrombi.

The onset of venous thromboembolism, including pulmonary embolism, represents a significant health burden affecting more than 1 million people annually worldwide. Current treatment options are based on anticoagulation, which is suboptimal for preventing further embolic events. In order to develop better treatments for thromboembolism, we sought to understand the structural and mechanical properties of blood clots and how this influences embolism in vivo. We developed a murine model in which fibrin γ-chain cross-linking by activated Factor XIII is eliminated (FGG3X) and applied methods to study thromboembolism at whole-body and organ levels. We show that FGG3X mice have a normal phenotype, with overall coagulation parameters and platelet aggregation and function largely unaffected, except for total inhibition of fibrin γ-chain cross-linking. Elimination of fibrin γ-chain cross-linking resulted in thrombi with reduced strength that were prone to fragmentation. Analysis of embolism in vivo using Xtreme optical imaging and light sheet microscopy demonstrated that the elimination of fibrin γ-chain cross-linking resulted in increased embolization without affecting clot size or lysis. Our findings point to a central previously unrecognized role for fibrin γ-chain cross-linking in clot stability. They also indirectly indicate mechanistic targets for the prevention of thrombosis through selective modulation of fibrin α-chain but not γ-chain cross-linking by activated Factor XIII to reduce thrombus size and burden, while maintaining clot stability and preventing embolism.

Model Lipid Membranes Assembled from Natural Plant Thylakoids into 2D Microarray Patterns as a Platform to Assess the Organization and Photophysics of Light-Harvesting Proteins.

Natural photosynthetic "thylakoid" membranes found in green plants contain a large network of light-harvesting (LH) protein complexes. Rearrangement of this photosynthetic machinery, laterally within stacked membranes called "grana", alters protein-protein interactions leading to changes in the energy balance within the system. Preparation of an experimentally accessible model system that allows the detailed investigation of these complex interactions can be achieved by interfacing thylakoid membranes and synthetic lipids into a template comprised of polymerized lipids in a 2D microarray pattern on glass surfaces. This paper uses this system to interrogate the behavior of LH proteins at the micro- and nanoscale and assesses the efficacy of this model. A combination of fluorescence lifetime imaging and atomic force microscopy reveals the differences in photophysical state and lateral organization between native thylakoid and hybrid membranes, the mechanism of LH protein incorporation into the developing hybrid membranes, and the nanoscale structure of the system. The resulting model system within each corral is a high-quality supported lipid bilayer that incorporates laterally mobile LH proteins. Photosynthetic activity is assessed in the hybrid membranes versus proteoliposomes, revealing that commonly used photochemical assays to test the electron transfer activity of photosystem II may actually produce false-positive results.

Friction between soft contacts at nanoscale on uncoated and protein-coated surfaces.

The understanding of friction on soft sliding biological surfaces at the nanoscale is poorly understood as hard interfaces are frequently used as model systems. Herein, we studied the influence of elastic modulus on the frictional properties of model surfaces at the nanoscale for the first time. We prepared model silicone-based elastomer surfaces with tuneable modulus ranging from hundreds of kPa to a few MPa, similar to those found in real biological surfaces, and employed atomic force microscopy to characterize their modulus, adhesion, and surface morphology. Consequently, we used friction force microscopy to investigate nanoscale friction in hard-soft and soft-soft contacts using spherical colloidal probes covered by adsorbed protein films. Unprecedented results from this study reveal that modulus of a surface can have a significant impact on the frictional properties of protein-coated surfaces with higher deformability leading to lower contact pressure and, consequently, decreased friction. These important results pave the way forward for designing new functional surfaces for serving as models of appropriate deformability to replicate the mechanical properties of the biological structures and processes for accurate friction measurements at nanoscale.

Probing the frictional properties of soft materials at the nanoscale.

The understanding of friction in soft materials is of increasing importance due to the demands of industries such as healthcare, biomedical, food and personal care, the incorporation of soft materials into technology, and in the study of interacting biological interfaces. Many of these processes occur at the nanoscale, but even at micrometer length scales there are fundamental aspects of tribology that remain poorly understood. With the advent of Friction Force Microscopy (FFM), there have been many fundamental insights into tribological phenomena at the atomic scale, such as 'stick-slip' and 'super-lubricity'. This review examines the growing field of soft tribology, the experimental aspects of FFM and its underlying theory. Moving to the nanoscale changes the contact mechanics which govern adhesive forces, which in turn play a pivotal role in friction, along with the deformation of the soft interface and dissipative phenomena. We examine recent progress and future prospects in soft nanotribology.

Potential of Manuka Honey as a Natural Polyelectrolyte to Develop Biomimetic Nanostructured Meshes With Antimicrobial Properties.

The use of antibiotics has been the cornerstone to prevent bacterial infections; however, the emergency of antibiotic-resistant bacteria is still an open challenge. This work aimed to develop a delivery system for treating soft tissue infections for: (1) reducing the released antimicrobial amount, preventing drug-related systemic side effects; (2) rediscovering the beneficial effects of naturally derived agents; and (3) preserving the substrate functional properties. For the first time, Manuka honey (MH) was proposed as polyelectrolyte within the layer-by-layer assembly. Biomimetic electrospun poly(ε-caprolactone) meshes were treated via layer-by-layer assembly to obtain a multilayered nanocoating, consisting of MH as polyanion and poly-(allylamine-hydrochloride) as polycation. Physicochemical characterization demonstrated the successful nanocoating formation. Different cell lines (human immortalized and primary skin fibroblasts, and primary endothelial cells) confirmed positively the membranes cytocompatibility, while bacterial tests using Gram-negative and Gram-positive bacteria demonstrated that the antimicrobial MH activity was dependent on the concentration used and strains tested.

Sub-Nanometer Thick Gold Nanosheets as Highly Efficient Catalysts.

2D metal nanomaterials offer exciting prospects in terms of their properties and functions. However, the ambient aqueous synthesis of atomically-thin, 2D metallic nanomaterials represents a significant challenge. Herein, freestanding and atomically-thin gold nanosheets with a thickness of only 0.47 nm (two atomic layers thick) are synthesized via a one-step aqueous approach at 20 °C, using methyl orange as a confining agent. Owing to the high surface-area-to-volume ratio, abundance of unsaturated atoms exposed on the surface and large interfacial areas arising from their ultrathin 2D nature, the as-prepared Au nanosheets demonstrate excellent catalysis performance in the model reaction of 4-nitrophenol reduction, and remarkable peroxidase-mimicking activity, which enables a highly sensitive colorimetric sensing of H2O2 with a detection limit of 0.11 × 10-6 m. This work represents the first fabrication of freestanding 2D gold with a sub-nanometer thickness, opens up an innovative pathway toward atomically-thin metal nanomaterials that can serve as model systems for inspiring fundamental advances in materials science, and holds potential across a wide region of applications.

Nanoscale Substrate Roughness Hinders Domain Formation in Supported Lipid Bilayers.

Supported lipid bilayers are model membranes formed at solid substrate surfaces. This architecture renders the membrane experimentally accessible to surface-sensitive techniques used to study their properties, including atomic force microscopy, optical fluorescence microscopy, quartz crystal microbalance, and X-ray/neutron reflectometry, and allows integration with technology for potential biotechnological applications such as drug screening devices. The experimental technique often dictates substrate choice or treatment, and it is anecdotally recognized that certain substrates are suitable for a particular experiment, but the exact influence of the substrate has not been comprehensively investigated. Here, we study the behavior of a simple model bilayer, phase-separating on a variety of commonly used substrates, including glass, mica, silicon, and quartz, with drastically different results. The distinct micron-scale domains observed on mica, identical to those seen in free-floating giant unilamellar vesicles, are reduced to nanometer-scale domains on glass and quartz. The mechanism for the arrest of domain formation is investigated, and the most likely candidate is nanoscale surface roughness, acting as a drag on the hydrodynamic motion of small domains during phase separation. Evidence was found that the physicochemical properties of the surface have a mediating effect, most likely because of the changes in the lubricating interstitial water layer between the surface and bilayer.

Proteoliposomes as energy transferring nanomaterials: enhancing the spectral range of light-harvesting proteins using lipid-linked chromophores.

Bio-hybrid nanomaterials have great potential for combining the most desirable aspects of biomolecules and the contemporary concepts of nanotechnology to create highly efficient light-harvesting materials. Light-harvesting proteins are optimized to absorb and transfer solar energy with remarkable efficiency but have a spectral range that is limited by their natural pigment complement. Herein, we present the development of model membranes ("proteoliposomes") in which the absorption range of the membrane protein Light-Harvesting Complex II (LHCII) is effectively enhanced by the addition of lipid-tethered Texas Red (TR) chromophores. Energy transfer from TR to LHCII is observed with up to 94% efficiency and increased LHCII fluorescence of up to three-fold when excited in the region of lowest natural absorption. The new self-assembly procedure offers the modularity to control the concentrations incorporated of TR and LHCII, allowing energy transfer and fluorescence to be tuned. Fluorescence Lifetime Imaging Microscopy provides single-proteoliposome-level quantification of energy transfer efficiency and confirms that functionality is retained on surfaces. Designer proteoliposomes could act as a controllable light-harvesting nanomaterial and are a promising step in the development of bio-hybrid light-harvesting systems.

Quantitative Analysis of Structure and Dynamics in AFM Images of Lipid Membranes.

AFM is now established as a powerful and direct technique for studying lipid membranes, and is highly complementary with other techniques. It is the only method for direct imaging and mechanical probing of lipid phase structure in a liquid environment down to the nanometer level. In order to understand the structure, function, and interactions of membranes at this level, we must be able to reliably and quantitatively measure the AFM images. Here we describe the methods used to process and analyze AFM images of phase-separated supported lipid bilayers . This initially takes a static approach, where we simply quantify the % of domain area, number of domains, and morphology, and quantify how many images must be taken to obtain reliable statistics. We then look at dynamics, describing the methods we use to study the nanometer scale motion of the domain perimeter as observed using Fast Scan AFM, and hence extract a quantitative line tension.

Interactions between callose and cellulose revealed through the analysis of biopolymer mixtures.

The properties of (1,3)-ß-glucans (i.e., callose) remain largely unknown despite their importance in plant development and defence. Here we use mixtures of (1,3)-ß-glucan and cellulose, in ionic liquid solution and hydrogels, as proxies to understand the physico-mechanical properties of callose. We show that after callose addition the stiffness of cellulose hydrogels is reduced at a greater extent than predicted from the ideal mixing rule (i.e., the weighted average of the individual components' properties). In contrast, yield behaviour after the elastic limit is more ductile in cellulose-callose hydrogels compared with sudden failure in 100% cellulose hydrogels. The viscoelastic behaviour and the diffusion of the ions in mixed ionic liquid solutions strongly indicate interactions between the polymers. Fourier-transform infrared analysis suggests that these interactions impact cellulose organisation in hydrogels and cell walls. We conclude that polymer interactions alter the properties of callose-cellulose mixtures beyond what it is expected by ideal mixing.

Nanoscale mechanics of microgel particles.

Microgel particles are highly tuneable materials that are useful for a wide range of industrial applications, such as drug delivery, sensing, nanoactuation, emulsion stabilisation and use as cell substrates. Microgels have also been used as model systems investigating physical phenomena such as crystallization, glass-formation, jamming, ageing and complex flow behaviour. The responsiveness of microgel systems such as poly(N-isopropylacrylamide) (PNIPAm) to external stimuli has been established in fundamental investigations and in applications and recent work has begun to quantify the mechanics of individual particles. However little focus has been placed on determining their internal mechanical properties, which is likely to relate to their nonuniform internal structure. In this work we combine atomic force microscopy, force spectroscopy and dynamic light scattering to mechanically profile the internal structure of microgel particles in the size range of ∼100 nm, which is commonly used both in practical applications and in fundamental studies. Nanoindentation using thermally stable cantilevers allows us to determine the particle moduli and the deformation profiles during particle compression with increasing force, while peak force nanomechanical mapping (PF-QNM) AFM is used to capture high resolution images of the particles' mechanical response. Combining these approaches with dynamic light scattering allows a quantitative profile of the particles' internal elastic response to be determined. Our results provide clear evidence for a radial distribution in particle mechanical response with a softer outer "corona" and a stiffer particle core. We determine the particle moduli in the core and corona, using different force microscopy approaches, and find them to vary systematically both in the core (∼17-50 kPa) and at the outer periphery of the particles (∼3-40 kPa). Importantly, we find that highly crosslinked particles have equivalent moduli across their radial profile, reflecting their significantly lower radial heterogeneity. This ability to accurately and precisely probe microgel radial profiles has clear implications both for fundamental science and for industrial applications.

A fibrin biofilm covers blood clots and protects from microbial invasion.

Hemostasis requires conversion of fibrinogen to fibrin fibers that generate a characteristic network, interact with blood cells, and initiate tissue repair. The fibrin network is porous and highly permeable, but the spatial arrangement of the external clot face is unknown. Here we show that fibrin transitioned to the blood-air interface through Langmuir film formation, producing a protective film confining clots in human and mouse models. We demonstrated that only fibrin is required for formation of the film, and that it occurred in vitro and in vivo. The fibrin film connected to the underlying clot network through tethering fibers. It was digested by plasmin, and formation of the film was prevented with surfactants. Functionally, the film retained blood cells and protected against penetration by bacterial pathogens in a murine model of dermal infection. Our data show a remarkable aspect of blood clotting in which fibrin forms a protective film covering the external surface of the clot, defending the organism against microbial invasion.