Characterizing and Tuning the Properties of Polydiacetylene Films for Sensing Applications.

Self-assembled, polymerized diacetylene (DA) nanostructures and two-dimensional films have been studied over the past two decades for sensor applications because of their straightforward visual readout. DA monomers, when exposed to UV light, polymerize to produce a visibly blue polymer. Blue phase polydiacetylenes (PDAs) when exposed to an external stimuli, such as temperature or UV light, undergo a chromatic phase transition to a fluorescent, visibly red phase. The tunability of the monomer to blue to red chromatic phase transitions by choice of diacetylene monomer in the presence of metal cations is systematically and comprehensively investigated to determine their effects on the properties of PDA Langmuir films. The polymerization kinetics and domain morphology of the PDA films were characterized using polarized fluorescent microscopy, UV-vis-fluorescent spectroscopy, and Fourier transform infrared spectroscopy (FTIR). Increasing the monomer alkyl tail length was found to strongly increase the UV dose necessary to produce optimally blue films and fully red films. A decrease in the polymer domain size was also correlated with longer-tailed DA molecules. Metal cations have a diverse effect on the film behavior. Alkaline-earth metals such as Mg, Ca, and Ba have a negligible effect on the phase transition kinetics but can be used to tune PDA polymer domain sizes. The Ni and Fe cations increase the UV dose necessary to produce red phase PDA films and significantly decrease the polymer domain sizes. The Zn, Cd, and Cu ions exhibit strong directed interactions with the PDA carboxylic acid headgroups, resulting in quenched fluorescence and a unique film morphology. FTIR analysis provides insight into the metal-PDA binding mechanisms and demonstrates that the coordination between the PDA film headgroups and the metal cations can be correlated with changes in the film morphology and kinetics. The findings from these studies will have broad utility for tuning PDA-based sensors for different applications and sensitivity ranges.

Direct Measurements of the Forces between Silver and Mica in Humic Substance-Rich Solutions.

Deposition of engineered nanoparticles onto porous media from flowing suspensions is important for soil and groundwater quality. The deposition mechanism is controlled by interaction forces between particles and collectors. We investigated the origin and magnitude of opposing forces between silver and mica surfaces (representing nanosilver and sand grains) in solutions relevant to agricultural soils with direct measurements using a surface force apparatus. Solutions of variable NaNO3, Ca(NO3)2, and humic acid (HA) concentrations were used to differentiate individual contributing forces and quantify surface properties. The measured Hamaker constant for silver-water-mica was consistent with Lifshitz theory. Our results indicate that HA forms an adsorbed surface layer, but its charge, thicknesses, compressibility, and mass are significantly larger on mica than silver. Ca2+ primarily reduced the differences between the initially adsorbed HA layer properties on each surface, making them more similar. Force-distance profiles indicate that, when silver-mica systems were exposed to HA, osmotic-steric, electrostatic, and van der Waals forces dominate. Soft particle theory was deemed inappropriate for this system. Derjaguin's approximation was utilized to translate force measurements into interaction energy between nanosilver particles and mica collectors. We propose attachment efficiency estimates from measured surface properties, which suggest high particle mobility when nanosilver is applied to HA-rich agricultural soils with modest ionic strength.

Melting-Temperature-Dependent Interactions of Ergosterol with Unsaturated and Saturated Lipids in Model Membranes.

Sterols such as cholesterol (Chol) and ergosterol (Erg) are known to regulate membrane properties in higher eukaryotes and in lower eukaryotes, respectively. To better understand the modulation of membrane properties by Erg, binary lipid membranes composed of Erg and diacylglycerophosphocholine (PC) were studied in Langmuir monolayer and bilayer vesicle systems. From the excess area measured by pressure-area isotherms, attractive interactions between Erg and saturated PC were significant above the melting temperature (Tm) of PC. Conversely, repulsive interactions were observed at temperatures below Tm. From the analyses of membrane fluidity and polarity using fluorescence probes, similar trends were observed for bilayer systems where Erg had an ordering effect on saturated PC vesicles in the fluid state. However, Chol had a stronger ordering effect than Erg. In unsaturated PC systems, Erg did not alter membrane ordering. These findings demonstrate that the interaction of Erg with the fluid-state PC lipids will maintain lower-eukaryote membranes in a more ordered state, similar to the effect of cholesterol in higher eukaryotes.

Lipid and Protein Transfer between Nanolipoprotein Particles and Supported Lipid Bilayers.

A nanolipoprotein particle (NLP) is a lipid bilayer disc stabilized by two amphipathic "scaffold" apolipoproteins. It has been most notably utilized as a tool for solubilizing a variety of membrane proteins while preserving structural and functional properties. Transfer of functional proteins from NLPs into model membrane systems such as supported lipid bilayers (SLBs) would enable new opportunities, for example, two-dimensional protein crystallization and studies on protein-protein interactions. This work used fluorescence microscopy and atomic force microscopy to investigate the interaction between NLPs and SLBs. When incubated with SLBs, NLPs were found to spontaneously deliver lipid and protein cargo. The impact of membrane composition on lipid exchange was explored, revealing a positive correlation between the magnitude of lipid transfer and concentration of defects in the target SLB. Incorporation of lipids capable of binding specifically to polyhistidine tags encoded into the apolipoproteins also boosted transfer of NLP cargo. Optimal conditions for lipid and protein delivery from NLPs to SLBs are proposed based on interaction mechanisms.

Mechanism of Acid-Triggered Cargo Release from Lipid Bilayer-Coated Mesoporous Silica Particles.

Lipid bilayer-coated mesoporous silica nanoparticles are unique core-shell nanomaterials currently being developed as drug delivery vehicles. To improve cargo loading and biocirculation, the pore structure and surface chemistry of the particle have been modified and well characterized. However, an understanding of cargo release mechanisms from cellular uptake pathways remains largely unexplored. Here, we present a study of the release mechanism of lipid bilayer-coated silica particles induced by endosomal-like pH change from 7.4 to 5.0. We found that this relatively small pH change produces rapid deformation of the supported lipid bilayer that ultimately results in holes in the membrane. Using a combination of dye release studies, wide-field and confocal fluorescence microscopies, and surface area modeling analysis, we determined that small blister-like structures are formed, which lead to lateral membrane displacement and hole formation. Possible mechanisms for the blister formation, which include curvature effects and interfacial interactions, are discussed.

Preparation and Characterization of Solid-Supported Lipid Bilayers Formed by Langmuir-Blodgett Deposition: A Tutorial.

The structure, phase behavior, and properties of cellular membranes are derived from their composition, which includes phospholipids, sphingolipids, sterols, and proteins with various levels of glycosylation. Because of the intricate nature of cellular membranes, a plethora of in vitro studies have been carried out with model membrane systems that capture particular properties such as fluidity, permeability, and protein binding but vastly simplify the membrane composition in order to focus in detail on a specialized property or function. Supported lipid bilayers (SLB) are widely used as archetypes for cellular membranes, and this instructional review primarily focuses on the preparation and characterization of SLB systems formed by Langmuir deposition methods. Typical characterization methods, which take advantage of the planar orientation of SLBs, are illustrated, and references that go into more depth are included. This invited instructional review is written so that nonexperts can quickly gain in-depth knowledge regarding the preparation and characterization of SLBs. In addition, this work goes beyond traditional instructional reviews to provide expert readers with new results that cover a wider range of SLB systems than those previously reported in the literature. The quality of an SLB is frequently not well described, and details such as topological defects can influence the results and conclusions of an individual study. This article quantifies and compares the quality of SLBs fabricated from a variety of gel and fluid compositions, in correlation with preparation techniques and parameters, to generate general rules of thumb to guide the construction of designed SLB systems.

New Algorithm to Enable Construction and Display of 3D Structures from Scanning Probe Microscopy Images Acquired Layer-by-Layer.

Scanning probe microscopy (SPM), such as atomic force microscopy (AFM), is widely known for high-resolution imaging of surface structures and nanolithography in two dimensions (2D), providing important physical insights into surface science and material science. This work reports a new algorithm to enable construction and display of layer-by-layer 3D structures from SPM images. The algorithm enables alignment of SPM images acquired during layer-by-layer deposition and removal of redundant features and faithfully constructs the deposited 3D structures. The display uses a "see-through" strategy to enable the structure of each layer to be visible. The results demonstrate high spatial accuracy as well as algorithm versatility; users can set parameters for reconstruction and display as per image quality and research needs. To the best of our knowledge, this method represents the first report to enable SPM technology for 3D imaging construction and display. The detailed algorithm is provided to facilitate usage of the same approach in any SPM software. These new capabilities support wide applications of SPM that require 3D image reconstruction and display, such as 3D nanoprinting and 3D additive and subtractive manufacturing and imaging.

Interaction forces and membrane charge tunability: Oleic acid containing membranes in different pH conditions.

Oleic acid is known to interact with saturated lipid molecules and increase the fluidity of gel phase lipid membranes. In this work, the thermodynamic properties of mixed monolayers of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and oleic acid at the air-water interface were determined using Langmuir isotherms. The isotherm study revealed an attractive interaction between oleic acid and DPPC. The incorporation of oleic acid also monotonically decreased the elastic modulus of the monolayer indicative of higher fluidity with increasing oleic acid content. Using the surface force apparatus, intermembrane force-distance profiles were obtained for substrate supported DPPC membranes containing 30mol% oleic acid at pH5.8 and 7.4. Three different preparation conditions resulted in distinct force profiles. Membranes prepared in pH5.8 subphase had a low number of nanoscopic defects ≤1% and an adhesion magnitude of ~0.6mN/m. A slightly higher defect density of 1-4% was found for membranes prepared in a physiological pH7.4 subphase. The presence of the exposed hydrophobic moieties resulted in a higher adhesion magnitude of 2.9mN/m. Importantly, at pH7.4, some oleic acid deprotonates resulting in a long-range electrostatic repulsion. Even though oleic acid increased the DPPC bilayer fluidity and the number of defects, no membrane restructuring was observed indicating that the system maintained a stable configuration.

Interaction Forces between Lipid Rafts.

Cellular membranes containing sphingolipids and cholesterol have been shown to self-organize into lipid rafts-specialized domains that host integral membrane proteins and modulate the bioactivity of cells. In this work, force-distance profiles between raft membranes in the liquid-ordered phase consisting of singly unsaturated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), a complex mixture of brain sphingomyelin (BSM), and cholesterol were measured using the surface force apparatus (SFA). Two distinct force profiles were detected corresponding to uniform raft membranes and raft membranes with a higher level of topological membrane defects (heterogeneous) as corroborated by atomic force microscopy (AFM) scans. In all cases a weak, long-range electrostatic repulsion was observed with some variation in the surface charge density. The variation in electrostatic repulsion was attributed to charged lipid species primarily from the constituent lipids in the BSM mixture. The adhesion between the uniform raft membranes was comparable to our previous work with pure component, liquid-ordered POPC-DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine)-cholesterol membranes. Raft membranes with more topological defects adhered more strongly owing to hydrophobic attraction between exposed acyl chains. Even though the rafts were in the liquid-ordered phase and membrane defects were present in the contact region, the raft membranes were stable, and no structural rearrangement was observed throughout the measurements. Our findings demonstrate that liquid-ordered membranes are stable to mechanical loading and not particularly sensitive to compositional variation.

Density and Phase State of a Confined Nonpolar Fluid.

Measurements of the mean refractive index of a spherelike nonpolar fluid, octamethytetracylclosiloxane (OMCTS), confined between mica sheets, demonstrate direct and conclusive experimental evidence of the absence of a first-order liquid-to-solid phase transition in the fluid when confined, which has been suggested to occur from previous experimental and simulation results. The results also show that the density remains constant throughout confinement, and that the fluid is incompressible. This, along with the observation of very large increases (many orders of magnitude) in viscosity during confinement from the literature, demonstrate that the molecular motion is limited by the confining wall and not the molecular packing. In addition, the recently developed refractive index profile correction method, which enables the structural perturbation inherent at a solid-liquid interface and that of a liquid in confinement to be determined independently, was used to show that there was no measurable excess or depleted mass of OMCTS near the mica surface in bulk films or confined films of only two molecular layers.

Characterization of solid-supported dipalmitoylphosphatidylcholine membranes containing cholesterol.

The incorporation of cholesterol into dipalmitoylphosphatidylcholine (DPPC) membranes, even in small amounts, has been shown to significantly alter the properties of the membrane. In this work, force-distance interaction profiles of DPPC membranes containing 8 mol % cholesterol obtained using the surface force apparatus are analyzed in the context of high-resolution structural characterization by atomic force microscopy and neutron reflectometry. The adhesion between the mixed membranes was greater than that for pure DPPC and was variable-depending on the number of defects in the outer membrane leaflets. These defects were only detectable by atomic force microscopy and had an average size of 230 ± 30 nm and 1-5% surface density in the outer leaflet. The adhesion between the membranes monotonically increased as the thickness of the membrane decreased-in direct correlation with the number of defects present (exposed hydrophobic groups) in the membrane contact region. Because of the low diffusion rate of gel-phase membranes, the interaction force profiles were stable and no membrane restructuring was observed.

Analyzing refractive index profiles of confined fluids by interferometry.

This work describes an interferometry data analysis method for determining the optical thickness of thin films or any variation in the refractive index of a fluid or film near a surface. In particular, the method described is applied to the analysis of interferometry data taken with a surface force apparatus (SFA). The technique does not require contacting or confining the fluid or film. By analyzing interferometry data taken at many intersurface separation distances out to at least 300 nm, the properties of a film can be quantitatively determined. The film can consist of material deposited on the surface, like a polymer brush, or variation in a fluid's refractive index near a surface resulting from, for example, a concentration gradient, depletion in density, or surface roughness. The method is demonstrated with aqueous polyethylenimine (PEI) adsorbed onto mica substrates, which has a large concentration and therefore refractive index gradient near the mica surface. The PEI layer thickness determined by the proposed method is consistent with the thickness measured by conventional SFA methods. Additionally, a thorough investigation of the effects of random and systematic error in SFA data analysis and modeling via simulations of interferometry is described in detail.

Interaction forces between ternary lipid bilayers containing cholesterol.

Interaction force-distance profiles between substrate-supported membranes composed of equimolar ternary mixtures of unsaturated phosphotidylcholine (PC) lipid, saturated PC lipid, and cholesterol were determined using the surface force apparatus. Both double and single unsaturated PC lipids were studied. In all cases, the membranes were slightly negatively charged, resulting in a weak, long-range electrostatic repulsion. Corroborative atomic force microscopy, zeta potential, and fluorescence microscopy measurements were used to establish that a small level of charged lipid impurities (∼1/400 lipid molecules) were responsible for the repulsive electrostatic interaction between the membranes. At contact, the membranes were adhesive. The magnitude of the adhesion was greater than the van der Waals interaction between pure PC membranes without cholesterol. The enhanced adhesion was primarily attributed to hydrophobic attraction due to the presence of nanoscopic membrane defects which exposed the underlying membrane leaflet. The interaction force-distance profiles also demonstrated that the nanoscopic defects enabled membrane restructuring in the contact region.

Thickness and refractive index of DPPC and DPPE monolayers by multiple-beam interferometry.

The thickness and refractive index of 1,2-dipalmitoyl-sn-glycero-3-phosphatidyl choline (DPPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) monolayers Langmuir--Blodgett (LB) deposited on mica were measured in dry air and bulk water using multiple-beam interferometry (MBI). Measurements of thickness using atomic force microscopy (AFM) of identical monolayers, and X-ray reflectivity (XRR) of the monolayers on quartz were taken for comparison. The measurement of the properties of solid-supported monolayers in dry air allows lipid optical properties to be determined free from solvent effects. The thickness and refractive index measured by MBI were 25.5 ± 0.6 Å and 1.485 ± 0.007 for DPPE monolayers, and 23.9 ± 0.5 Å and 1.478 ± 0.006 for DPPC monolayers in dry air. These thicknesses are consistent with the other techniques used in this work as well as other measurements in the literature. The refractive indices of solid-supported lipid monolayers have not been previously measured. The values are higher than previous measurements on black lipid films done by reflectometry, which is attributed to increased lipid packing density and the absence of hydrocarbon solvents. Applying water to the monolayers had no measurable effect on their properties, indicating that any change in hydration was below detection.

Interaction forces between DPPC bilayers on glass.

The surface force apparatus (SFA) was utilized to obtain force-distance profiles between silica-supported membranes formed by Langmuir-Blodgett deposition of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). In the absence of a membrane, a long-range electrostatic repulsion and short-range steric repulsion are measured as a result of the deprotonation of silica in water and the roughness of the silica film. The electrostatic repulsion is partially screened by the lipid membrane, and a van der Waals adhesion comparable to that measured with well-packed DPPC membranes on mica is measured. This finding suggest that electrostatic interactions due to the underlying negatively charged silica are likely present in other systems of glass-supported membranes. In contrast, the charge of an underlying mica substrate is almost completely screened when a lipid membrane is deposited on the mica. The difference in the two systems is attributed to the stronger physisorption of zwitterionic lipids to molecularly smooth mica compared to physisorption to rougher silica.

Tracking Mechanical Stress and Cell Migration with Inexpensive Polymer Thin-Film Sensors.

Polydiacetylene (PDA) Langmuir films are well known for their blue to red chromatic transitions in response to a variety of stimuli, including UV light, heat, bio-molecule bindings and mechanical stress. In this work, we detail the ability to tune PDA Langmuir films to exhibit discrete chromatic transitions in response to applied mechanical stress. Normal and shear-induced transitions were quantified using the Surface Forces Apparatus and established to be binary and tunable as a function of film formation conditions. Both monomer alkyl tail length and metal cations were used to manipulate the chromatic transition force threshold to enable discrete force sensing from ~50 to ~500 nN µm-2 for normal loading and ~2 to ~40 nN µm-2 for shear-induced transitions, which are appropriate for biological cells. The utility of PDA thin-film sensors was demonstrated with the slime mold Physarum polycephalum. The fluorescence readout of the films enabled: the area explored by Physarum to be visualized, the forces involved in locomotion to be quantified, and revealed novel puncta formation potentially associated with Physarum sampling its environment.

All polymer microfluidic chips-A fixed target sample delivery workhorse for serial crystallography.

The development of x-ray free electron laser (XFEL) light sources and serial crystallography methodologies has led to a revolution in protein crystallography, enabling the determination of previously unobtainable protein structures and near-atomic resolution of otherwise poorly diffracting protein crystals. However, to utilize XFEL sources efficiently demands the continuous, rapid delivery of a large number of difficult-to-handle microcrystals to the x-ray beam. A recently developed fixed-target system, in which crystals of interest are enclosed within a sample holder, which is rastered through the x-ray beam, is discussed in detail in this Perspective. The fixed target is easy to use, maintains sample hydration, and can be readily modified to allow a broad range of sample types and different beamline requirements. Recent innovations demonstrate the potential of such microfluidic-based fixed targets to be an all-around "workhorse" for serial crystallography measurements. This Perspective will summarize recent advancements in microfluidic fixed targets for serial crystallography, examine needs for future development, and guide users in designing, choosing, and utilizing a fixed-target sample delivery device for their system.

A user-friendly plug-and-play cyclic olefin copolymer-based microfluidic chip for room-temperature, fixed-target serial crystallography.

Over the past two decades, serial X-ray crystallography has enabled the structure determination of a wide range of proteins. With the advent of X-ray free-electron lasers (XFELs), ever-smaller crystals have yielded high-resolution diffraction and structure determination. A crucial need to continue advancement is the efficient delivery of fragile and micrometre-sized crystals to the X-ray beam intersection. This paper presents an improved design of an all-polymer microfluidic `chip' for room-temperature fixed-target serial crystallography that can be tailored to broadly meet the needs of users at either synchrotron or XFEL light sources. The chips are designed to be customized around different types of crystals and offer users a friendly, quick, convenient, ultra-low-cost and robust sample-delivery platform. Compared with the previous iteration of the chip [Gilbile et al. (2021), Lab Chip, 21, 4831-4845], the new design eliminates cleanroom fabrication. It has a larger imaging area to volume, while maintaining crystal hydration stability for both in situ crystallization or direct crystal slurry loading. Crystals of two model proteins, lysozyme and thaumatin, were used to validate the effectiveness of the design at both synchrotron (lysozyme and thaumatin) and XFEL (lysozyme only) facilities, yielding complete data sets with resolutions of 1.42, 1.48 and 1.70 Å, respectively. Overall, the improved chip design, ease of fabrication and high modifiability create a powerful, all-around sample-delivery tool that structural biologists can quickly adopt, especially in cases of limited sample volume and small, fragile crystals.

Impact of membrane fluidity on steric stabilization by lipopolymers.

In this work, the impact of lipid lateral mobility on the steric interaction between membranes containing poly(ethylene glycol) (PEG) functionalized lipids was investigated using the surface force apparatus. The force-distance profiles show the presence of electrostatic and steric repulsion that arise from the presence of negatively charged PEG functionalized lipids. Fluid-phase bilayers have high lateral diffusion relative to gel-phase bilayers; however, a quantitative comparison of the interaction forces between membranes in these two different phase states demonstrates a reduced rate of diffusion in the fluid phase for the PEG-lipids under constrained geometries. Thus, the amount of polymer in the contact zone can be modulated and is reduced with fluid membranes; however, complete exclusion was not achieved. As a result, the steric repulsion afforded by PEG chains or binding affinity of ligated PEG chains can only be modestly tailored by the phase state of the liposome.