Ions Adsorbed at Amorphous Solid/Solution Interfaces Form Wigner Crystal-like Structures.

When a surface is immersed in a solution, it usually acquires a charge, which attracts counterions and repels co-ions to form an electrical double layer. The ions directly adsorbed to the surface are referred to as the Stern layer. The structure of the Stern layer normal to the interface was described decades ago, but the lateral organization within the Stern layer has received scant attention. This is because instrumental limitations have prevented visualization of the ion arrangements except for atypical, model, crystalline surfaces. Here, we use high-resolution amplitude modulated atomic force microscopy (AFM) to visualize in situ the lateral structure of Stern layer ions adsorbed to polycrystalline gold, and amorphous silica and gallium nitride (GaN). For all three substrates, when the density of ions in the layer exceeds a system-dependent threshold, correlation effects induce the formation of close packed structures akin to Wigner crystals. Depending on the surface and the ions, the Wigner crystal-like structure can be hexagonally close packed, cubic, or worm-like. The influence of the electrolyte concentration, species, and valence, as well as the surface type and charge, on the Stern layer structures is described. When the system parameters are changed to reduce the Stern layer ion surface excess below the threshold value, Wigner crystal-like structures do not form and the Stern layer is unstructured. For gold surfaces, molecular dynamics (MD) simulations reveal that when sufficient potential is applied to the surface, ion clusters form with dimensions similar to the Wigner crystal-like structures in the AFM images. The lateral Stern layer structures presented, and in particular the Wigner crystal-like structures, will influence diverse applications in chemistry, energy storage, environmental science, nanotechnology, biology, and medicine.

Quantitative Detection of Biological Nanovesicles in Drops of Saliva Using Microcantilevers.

Extracellular nanovesicles (EVs) are lipid-based vesicles secreted by cells and are present in all bodily fluids. They play a central role in communication between distant cells and have been proposed as potential indicators for the early detection of a wide range of diseases, including different types of cancer. However, reliable quantification of a specific subpopulation of EVs remains challenging. The process is typically lengthy and costly and requires purification of relatively large quantities of biopsy samples. Here, we show that microcantilevers operated with sufficiently small vibration amplitudes can successfully quantify a specific subpopulation of EVs directly from a drop (0.1 mL) of unprocessed saliva in less than 20 min. Being a complex fluid, saliva is highly non-Newtonian, normally precluding mechanical sensing. With a combination of standard rheology and microrheology, we demonstrate that the non-Newtonian properties are scale-dependent, enabling microcantilever measurements with a sensitivity identical to that in pure water when operating at the nanoscale. We also address the problem of unwanted sensor biofouling by using a zwitterionic coating, allowing efficient quantification of EVs at concentrations down to 0.1 µg/mL, based on immunorecognition of the EVs' surface proteins. We benchmark the technique on model EVs and illustrate its potential by quantifying populations of natural EVs commonly present in human saliva. The method effectively bypasses the difficulty of targeted detection in non-Newtonian fluids and could be used for various applications, from the detection of EVs and viruses in bodily fluids to the detection of molecular clusters or nanoparticles in other complex fluids.

Simultaneous quantification of Young's modulus and dispersion forces with nanoscale spatial resolution.

Many advances in polymers and layered materials rely on a precise understanding of the local interactions between adjacent molecular or atomic layers. Quantifying dispersion forces at the nanoscale is particularly challenging with existing methods often time consuming, destructive, relying on surface averaging or requiring bespoke equipment. Here, we present a non-invasive method able to quantify the local mechanical and dispersion properties of a given sample with nanometer lateral precision. The method, based on atomic force microscopy (AFM), uses the frequency shift of a vibrating AFM cantilever in combination with established contact mechanics models to simultaneously derive the Hamaker constant and the effective Young's modulus at a given sample location. The derived Hamaker constant and Young's modulus represent an average over a small (typically <100) number of molecules or atoms. The oscillation amplitude of the vibrating AFM probe is used to select the length-scale of the features to analyse, with small vibrations able to resolve the contribution of sub-nanometric defects and large ones exploring effectively homogeneous areas. The accuracy of the method is validated on a range of 2D materials in air and water as well as on polymer thin films. We also provide the first experimental measurements of the Hamaker constant of HBN, MoT2, WSe2and polymer films, verifying theoretical predictions and computer simulations. The simplicity and robustness of the method, implemented with a commercial AFM, may support a broad range of technological applications in the growing field of polymers and nanostructured materials where a fine control of the van der Waals interactions is crucial to tune their properties.

Lipid bilayer fluidity and degree of order regulates small EVs adsorption on model cell membrane.

Small extracellular vesicles (sEVs) are known to play an important role in the communication between distant cells and to deliver biological information throughout the body. To date, many studies have focused on the role of sEVs characteristics such as cell origin, surface composition, and molecular cargo on the resulting uptake by the recipient cell. Yet, a full understanding of the sEV fusion process with recipient cells and in particular the role of cell membrane physical properties on the uptake are still lacking. Here we explore this problem using sEVs from a cellular model of triple-negative breast cancer fusing to a range of synthetic planar lipid bilayers both with and without cholesterol, and designed to mimic the formation of 'raft'-like nanodomains in cell membranes. Using time-resolved Atomic Force Microscopy we were able to track the sEVs interaction with the different model membranes, showing the process to be strongly dependent on the local membrane fluidity. The strongest interaction and fusion is observed over the less fluid regions, with sEVs even able to disrupt ordered domains at sufficiently high cholesterol concentration. Our findings suggest the biophysical characteristics of recipient cell membranes to be crucial for sEVs uptake regulation.

Nanoscale probing of local dielectric changes at the interface between solids and aqueous saline solutions.

The mobility of dissolved ions and charged molecules at interfaces underpins countless processes in science and technology. Experimentally, this is typically measured from the averaged response of the charges to an electrical potential. High-resolution Atomic Force Microscopy (AFM) can image single adsorbed ions and molecules at solid-liquid interfaces, but probing the associated dynamics remains highly challenging. One possible strategy is to investigate the response of the species of interest to a highly localized AC electric field in an approach analogous to dielectric spectroscopy. The dielectric force experienced by the AFM tip apex is modulated by the dielectric properties of the sample probed, itself sensitive to the mobilities of solvated charges and dipoles. Previous work successfully used this approach to quantify the dielectric constant of thin samples, but with limited spatial resolution. Here we propose a strategy to simultaneously map the nanoscale topography and local dielectric variations across a range of interfaces by conducting high-resolution AFM imaging concomitantly with electrical AC measurements in a multifrequency approach. The strategy is tested over a 500 MHz bandwidth in pure liquids with different dielectric constants and in saline aqueous solutions. In liquids with higher dielectric constants, the system behaves as inductive-resistive-capacitive but the adjunction of ions removes the inductive resonances and precludes measurements at higher frequencies. High-resolution imaging is demonstrated over single graphene oxide (GrO) flakes with simultaneous but decoupled dielectric measurements. The dielectric constant is consistent and reproducible across liquids, except at higher salt concentrations where frequency-dependent effects occur. The results suggest the strategy is suitable for nanometre-level mapping of the dielectric properties of solid-liquid interfaces, but more work is needed to fully understand the different physical effects underpinning the measurements.

Hydroxide films on mica form charge-stabilized microphases that circumvent nucleation barriers.

Crystal nucleation is facilitated by transient, nanoscale fluctuations that are extraordinarily difficult to observe. Here, we use high-speed atomic force microscopy to directly observe the growth of an aluminum hydroxide film from an aqueous solution and characterize the dynamically fluctuating nanostructures that precede its formation. Nanoscale cluster distributions and fluctuation dynamics show many similarities to the predictions of classical nucleation theory, but the cluster energy landscape deviates from classical expectations. Kinetic Monte Carlo simulations show that these deviations can arise from electrostatic interactions between the clusters and the underlying substrate, which drive microphase separation to create a nanostructured surface phase. This phase can evolve seamlessly from a low-coverage state of fluctuating clusters into a high-coverage nanostructured network, allowing the film to grow without having to overcome classical nucleation barriers.

Behavior of Citrate-Capped Ultrasmall Gold Nanoparticles on a Supported Lipid Bilayer Interface at Atomic Resolution.

Nanomaterials have the potential to transform biological and biomedical research, with applications ranging from drug delivery and diagnostics to targeted interference of specific biological processes. Most existing research is aimed at developing nanomaterials for specific tasks such as enhanced biocellular internalization. However, fundamental aspects of the interactions between nanomaterials and biological systems, in particular, membranes, remain poorly understood. In this study, we provide detailed insights into the molecular mechanisms governing the interaction and evolution of one of the most common synthetic nanomaterials in contact with model phospholipid membranes. Using a combination of atomic force microscopy (AFM) and molecular dynamics (MD) simulations, we elucidate the precise mechanisms by which citrate-capped 5 nm gold nanoparticles (AuNPs) interact with supported lipid bilayers (SLBs) of pure fluid (DOPC) and pure gel-phase (DPPC) phospholipids. On fluid-phase DOPC membranes, the AuNPs adsorb and are progressively internalized as the citrate capping of the NPs is displaced by the surrounding lipids. AuNPs also interact with gel-phase DPPC membranes where they partially embed into the outer leaflet, locally disturbing the lipid organization. In both systems, the AuNPs cause holistic perturbations throughout the bilayers. AFM shows that the lateral diffusion of the particles is several orders of magnitude smaller than that of the lipid molecules, which creates some temporary scarring of the membrane surface. Our results reveal how functionalized AuNPs interact with differing biological membranes with mechanisms that could also have implications for cooperative membrane effects with other molecules.

Development of a flexure-based nano-actuator for high-frequency high-resolution directional sensing with atomic force microscopy.

Scanning probe microscopies typically rely on the high-precision positioning of a nanoscale probe in order to gain local information about the properties of a sample. At a given location, the probe is used to interrogate a minute region of the sample, often relying on dynamical sensing for improved accuracy. This is the case for most force-based measurements in atomic force microscopy (AFM) where sensing occurs with a tip oscillating vertically, typically in the kHz to MHz frequency regime. While this approach is ideal for many applications, restricting dynamical sensing to only one direction (vertical) can become a serious limitation when aiming to quantify the properties of inherently three-dimensional systems, such as a liquid near a wall. Here, we present the design, fabrication, and calibration of a miniature high-speed scanner able to apply controlled fast and directional in-plane vibrations with sub-nanometer precision. The scanner has a resonance frequency of ∼35 kHz and is used in conjunction with a traditional AFM to augment the measurement capabilities. We illustrate its capabilities at a solid-liquid interface where we use it to quantify the preferred lateral flow direction of the liquid around every sample location. The AFM can simultaneously acquire high-resolution images of the interface, which can be superimposed with the directional measurements. Examples of sub-nanometer measurements conducted with the new scanner are also presented.

Real-time tracking of ionic nano-domains under shear flow.

The behaviour of ions at solid-liquid interfaces underpins countless phenomena, from the conduction of nervous impulses to charge transfer in solar cells. In most cases, ions do not operate as isolated entities, but in conjunction with neighbouring ions and the surrounding solution. In aqueous solutions, recent studies suggest the existence of group dynamics through water-mediated clusters but results allowing direct tracking of ionic domains with atomic precision are scarce. Here, we use high-speed atomic force microscopy to track the evolution of Rb+, K+, Na+ and Ca2+ nano-domains containing 20 to 120 ions adsorbed at the surface of mica in aqueous solution. The interface is exposed to a shear flow able to influence the lateral motion of single ions and clusters. The results show that, when in groups, metal ions tend to move with a relatively slow dynamics, as can be expected from a correlated group motion, with an average residence timescale of ~ 1-2 s for individual ions at a given atomic site. The average group velocity of the clusters depends on the ions' charge density and can be explained by the ion's hydration state. The lateral shear flow of the fluid is insufficient to desorb ions, but indirectly influences the diffusion dynamics by acting on ions in close vicinity to the surface. The results provide insights into the dynamics of ion clusters when adsorbed onto an immersed solid under shear flow.

Impact of water on the lubricating properties of hexadecane at the nanoscale.

Fluid lubricants are routinely used to reduce friction in a wide range of applications, from car engines to machinery and hard-disk drives. However, their efficiency can be significantly influenced by the ambient conditions they are exposed to, in particular humidity. Our understanding of the molecular mechanisms responsible for the well-documented impact of water on lubrication remains limited, hindering the improvement of tribological formulations. Here, we use Atomic Force Microscopy (AFM) and shear force spectroscopy to investigate the structural and dynamical behaviour of a model lubricant, hexadecane, confined between an AFM probe and a hydrophilic mica surface at different temperatures and humidities. We show that both the nanoscale structure and the tribological behaviour of the system are dominated by the nucleation of water nanodroplets at the interface. The process is favoured at higher temperature and can be explained with classical nucleation theory whereby the droplets become stable when larger than 20 nm to 50 nm size, depending on the ambient conditions. Below this threshold, a molecularly thin film of water molecules coats the surface uniformly. Highly localised shear measurements demonstrate a detrimental impact of the nanodroplets on shear with a twofold increase in the lubricated friction force. However, this can be mitigated by the adjunction of an amphiphilic additive, here oleic acid.

Cotranscriptional Folding of a Bio-orthogonal Fluorescent Scaffolded RNA Origami.

The scaffolded origami technique is an attractive tool for engineering nucleic acid nanostructures. This paper demonstrates scaffolded RNA origami folding in vitro in which, for the first time, all components are transcribed simultaneously in a single-pot reaction. Double-stranded DNA sequences are transcribed by T7 RNA polymerase into scaffold and staple strands able to correctly fold in a high synthesis yield into the nanoribbon. Synthesis is successfully confirmed by atomic force microscopy, and the unpurified transcription reaction mixture is analyzed by an in gel-imaging assay where the transcribed RNA nanoribbons are able to capture the specific dye through the reconstituted split Broccoli aptamer showing a clear green fluorescent band. Finally, we simulate the RNA origami in silico using the nucleotide-level coarse-grained model oxRNA to investigate the thermodynamic stability of the assembled nanostructure in isothermal conditions over a period of time. Our work suggests that the scaffolded origami technique is a viable, and potentially more powerful, assembly alternative to the single-stranded origami technique for future in vivo applications.

Lubricated friction around nanodefects.

The lubrication properties of nanoconfined liquids underpin countless natural and industrial processes. However, our current understanding of lubricated friction is still limited, especially for nonideal interfaces exhibiting nanoscale chemical and topographical defects. Here, we use atomic force microscopy to explore the equilibrium and dynamical behavior of a model lubricant, squalane, confined between a diamond tip and graphite in the vicinity of an atomic step. We combine high-resolution imaging of the interface with highly localized shear measurements at different velocities and temperatures to derive a quantitative picture of the lubricated friction around surface defects. We show that defects tend to promote local molecular order and increase friction forces by reducing the number of stable molecular configurations in their immediate vicinity. The effect is general, can propagate over hundreds of nanometers, and can be quantitatively described by a semiempirical model that bridges the molecular details and mesoscale observations.

Coating and Stabilization of Liposomes by Clathrin-Inspired DNA Self-Assembly.

The self-assembly of the protein clathrin on biological membranes facilitates essential processes of endocytosis and has provided a source of inspiration for materials design by the highly ordered structural appearance. By mimicking the architecture of the protein building blocks and clathrin self-assemblies to coat liposomes with biomaterials, advanced hybrid carriers can be derived. Here, we present a method for fabricating DNA-coated liposomes by hydrophobically anchoring and subsequently connecting DNA-based triskelion structures on the liposome surface inspired by the assembly of the protein clathrin. Dynamic light scattering, ζ-potential, confocal microscopy, and cryo-electron microscopy measurements independently demonstrate successful DNA coating. Nanomechanical measurements conducted with atomic force microscopy show that the DNA coating enhances the mechanical stability of the liposomes relative to uncoated ones. Furthermore, we provide the possibility to reverse the coating process by triggering the disassembly of the DNA coats through a toehold-mediated displacement reaction. Our results describe a straightforward, versatile, and reversible approach for coating and stabilizing lipid vesicles through the assembly of rationally designed DNA structures. This method has potential for further development toward the ordered arrangement of tailored functionalities on the surface of liposomes and for applications as hybrid nanocarriers.

Long-lived ionic nano-domains can modulate the stiffness of soft interfaces.

Metal ions underpin countless processes at bio-interfaces, including maintaining electroneutrality, modifying mechanical properties and driving bioenergetic activity. These processes are typically described by ions behaving as independently diffusing point charges. Here we show that Na+ and K+ ions instead spontaneously form correlated nanoscale networks that evolve over seconds at the interface with an anionic bilayer in solution. Combining single-ion level atomic force microscopy and molecular dynamic simulations we investigate the configuration and dynamics of Na+, K+, and Rb+ at the lipid surface. We identify two distinct ionic states: the well-known direct electrostatic interaction with lipid headgroups and a water-mediated interaction that can drive the formation of remarkably long-lived ionic networks which evolve over many seconds. We show that this second state induces ionic network formation via correlative ion-ion interactions that generate an effective energy well of -0.4kBT/ion. These networks locally reduce the stiffness of the membrane, providing a spontaneous mechanism for tuning its mechanical properties with nanoscale precision. The ubiquity of water-mediated interactions suggest that our results have far-reaching implications for controlling the properties of soft interfaces.

Substrate-led cholesterol extraction from supported lipid membranes.

The lipid membrane is a principal building block in biology, technology and industry, where it often occurs supported by other hydrophilic structures. Interactions with the support can affect the physical behavior of the membrane from the local organization and diffusion of lipids and proteins, to phase transitions, and the local mechanical properties. In this study we show that supporting substrates textured with nanoscale hydrophilic and hydrophobic domains can modify the membrane's chemical composition by selectively extracting cholesterol molecules without affecting the remaining phospholipids. Using polydimethylsiloxane (PDMS) substrates with various degrees of plasma oxidation, we are able to trigger dramatic changes in the membrane morphology and biophysical properties, and relate them to the amount of extracted cholesterol. We also show that it is possible to control the cholesterol extraction through mechanical extension of the flexible PDMS support. Given the ubiquity of bio-substrates with textured surface properties and the wide use of PDMS we expect that our results will have implications not only in biological and chemical sciences but also in nanotechnologies such as organ on a chip technologies, biosensors, and stretchable bio-electronics.

Impact of Electric Fields on the Nanoscale Behavior of Lipid Monolayers at the Surface of Graphite in Solution.

The nanoscale organization and dynamics of lipid molecules in self-assembled membranes is central to the biological function of cells and in the technological development of synthetic lipid structures as well as in devices such as biosensors. Here, we explore the nanoscale molecular arrangement and dynamics of lipids assembled in monolayers at the surface of highly ordered pyrolytic graphite (HOPG), in different ionic solutions, and under electrical potentials. Using a combination of atomic force microscopy and fluorescence recovery after photobleaching, we show that HOPG is able to support fully formed and fluid lipid membranes, but mesoscale order and corrugations can be observed depending on the type of the lipid considered (1,2-dioleoyl- sn-glycero-3-phosphocholine, 1,2-dioleoyl- sn-glycero-3-phospho-l-serine (DOPS), and 1,2-dioleoyl-3-trimethylammoniumpropane) and the ion present (Na+, Ca2+, Cl-). Interfacial solvation forces and ion-specific effects dominate over the electrostatic changes induced by moderate electric fields (±1.0 V vs Ag/AgCl reference electrode) with particularly marked effects in the presence of calcium, and for DOPS. Our results provide insights into the interplay between the molecular, ionic, and electrostatic interactions and the formation of dynamical ordered structures in fluid lipid membranes.

Lubricating properties of single metal ions at interfaces.

The behaviour of ionic solutions confined in nanoscale gaps is central to countless processes, from biomolecular function to electrochemistry, energy storage and lubrication. However, no clear link exists between the molecular-level behaviour of the liquid and macroscopic observations. The problem mainly comes from the difficulty to interrogate a small number of liquid molecules. Here, we use atomic force microscopy to investigate the viscoelastic behaviour of pure water and ionic solutions down to the single ion level. The results show a glassy-like behaviour for pure water, with single metal ions acting as lubricants by reducing the elasticity of the nano-confined solution and the magnitude of the hydrodynamic friction. At small ionic concentration (<20 mM) the results can be quantitatively explained by the ions moving via a thermally-activated process resisted by the ion's hydration water (Prandtl-Tomlinson model). The model breaks down at higher salt concentrations due to ion-ion interaction effects that can no longer be neglected. The correlations are confirmed by direct sub-nanometre imaging of the interface at equilibrium. The results provide a molecular-level basis for explaining the tribological properties of aqueous solutions and suggest that ion-ion interactions create mesoscale effects that prevent a direct link between nanoscale and macroscopic measurements.

Isothermal folding of a light-up bio-orthogonal RNA origami nanoribbon.

RNA presents intringuing roles in many cellular processes and its versatility underpins many different applications in synthetic biology. Nonetheless, RNA origami as a method for nanofabrication is not yet fully explored and the majority of RNA nanostructures are based on natural pre-folded RNA. Here we describe a biologically inert and uniquely addressable RNA origami scaffold that self-assembles into a nanoribbon by seven staple strands. An algorithm is applied to generate a synthetic De Bruijn scaffold sequence that is characterized by the lack of biologically active sites and repetitions larger than a predetermined design parameter. This RNA scaffold and the complementary staples fold in a physiologically compatible isothermal condition. In order to monitor the folding, we designed a new split Broccoli aptamer system. The aptamer is divided into two nonfunctional sequences each of which is integrated into the 5' or 3' end of two staple strands complementary to the RNA scaffold. Using fluorescence measurements and in-gel imaging, we demonstrate that once RNA origami assembly occurs, the split aptamer sequences are brought into close proximity forming the aptamer and turning on the fluorescence. This light-up 'bio-orthogonal' RNA origami provides a prototype that can have potential for in vivo origami applications.