In Situ Atomic-Scale Imaging of Interfacial Water under 3D Nanoscale Confinement.

Capillary condensation of water from vapor is an everyday phenomenon which has a wide range of scientific and technological implications. Many aspects of capillary condensation are not well understood such as the structure of interfacial water, the existence of distinct properties of confined water, or the validity of the Kelvin equation at nanoscale. We note the absence of high-spatial resolution images inside a meniscus. Here, we develop an AFM-based method to provide in situ atomic-scale resolution maps of the solid-water interface of a nanomeniscus (80-250 nm3). The separation between the first two hydration layers on graphite is 0.30 nm, while on mica it is 0.28 nm. Those values are very close to the ones expected for the same surfaces immersed in bulk water. Thus, the hydration layer structure on a crystalline surface is independent of the water volume.

Tip Charge Dependence of Three-Dimensional AFM Mapping of Concentrated Ionic Solutions.

A molecular scale understanding of the organization and structure of a liquid near a solid surface is currently a major challenge in surface science. It has implications across different fields from electrochemistry and energy storage to molecular biology. Three-dimensional AFM generates atomically resolved maps of solid-liquid interfaces. The imaging mechanism behind those maps is under debate, in particular, for concentrated ionic solutions. Theory predicts that the observed contrast should depend on the tip's charged state. Here, by using neutrally, negatively, and positively charged tips, we demonstrate that the 3D maps depend on the tip's polarization. A neutral tip will explore the total particle density distribution (water and ions) while a charged tip will reveal the charge density distribution. The experimental data reproduce the key findings of the theory.

Molecular Recognition by Silicon Nanowire Field-Effect Transistor and Single-Molecule Force Spectroscopy.

Silicon nanowire (SiNW) field-effect transistors (FETs) have been developed as very sensitive and label-free biomolecular sensors. The detection principle operating in a SiNW biosensor is indirect. The biomolecules are detected by measuring the changes in the current through the transistor. Those changes are produced by the electrical field created by the biomolecule. Here, we have combined nanolithography, chemical functionalization, electrical measurements and molecular recognition methods to correlate the current measured by the SiNW transistor with the presence of specific molecular recognition events on the surface of the SiNW. Oxidation scanning probe lithography (o-SPL) was applied to fabricate sub-12 nm SiNW field-effect transistors. The devices were applied to detect very small concentrations of proteins (500 pM). Atomic force microscopy (AFM) single-molecule force spectroscopy (SMFS) experiments allowed the identification of the protein adsorption sites on the surface of the nanowire. We detected specific interactions between the biotin-functionalized AFM tip and individual avidin molecules adsorbed to the SiNW. The measurements confirmed that electrical current changes measured by the device were associated with the deposition of avidin molecules.

Interfacial layering of hydrocarbons on pristine graphite surfaces immersed in water.

Interfacial water participates in a wide range of phenomena involving graphite, graphite-like and 2D material interfaces. Recently, several high-spatial resolution experiments have questioned the existence of hydration layers on graphite, graphite-like and 2D material surfaces. Here, 3D AFM was applied to follow in real-time and with atomic-scale depth resolution the evolution of graphite-water interfaces. Pristine graphite surfaces upon immersion in water showed the presence of several hydration layers separated by a distance of 0.3 nm. Those layers were short-lived. After several minutes, the interlayer distance increased to 0.45 nm. At longer immersion times (∼50 min) we observed the formation of a third layer. An interlayer distance of 0.45 nm characterizes the layering of predominantly alkane-like hydrocarbons. Molecular dynamics calculations supported the experimental observations. The replacement of water molecules by hydrocarbons on graphite is spontaneous. It happens whenever the graphite-water volume is exposed to air.

Atomically resolved interfacial water structures on crystalline hydrophilic and hydrophobic surfaces.

Hydration layers are formed on hydrophilic crystalline surfaces immersed in water. Their existence has also been predicted for hydrophobic surfaces, yet the experimental evidence is controversial. Using 3D-AFM imaging, we probed the interfacial water structure of hydrophobic and hydrophilic surfaces with atomic-scale spatial resolution. We demonstrate that the atomic-scale structure of interfacial water on crystalline surfaces presents two antagonistic arrangements. On mica, a common hydrophilic crystalline surface, the interface is characterized by the formation of 2 to 3 hydration layers separated by approximately 0.3 nm. On hydrophobic surfaces such as graphite or hexagonal boron nitride (h-BN), the interface is characterized by the formation of 2 to 4 layers separated by about 0.5 nm. The latter interlayer distance indicates that water molecules are expelled from the vicinity of the surface and replaced by hydrocarbon molecules. This creates a new 1.5-2 nm thick interface between the hydrophobic surface and the bulk water. Molecular dynamics simulations reproduced the experimental data and confirmed the above interfacial water structures.

High-Speed Nanomechanical Mapping of the Early Stages of Collagen Growth by Bimodal Force Microscopy.

High-speed atomic force microscopy (AFM) enabled the imaging of protein interactions with millisecond time resolutions (10 fps). However, the acquisition of nanomechanical maps of proteins is about 100 times slower. Here, we developed a high-speed bimodal AFM that provided high-spatial resolution maps of the elastic modulus, the loss tangent, and the topography at imaging rates of 5 fps. The microscope was applied to identify the initial stages of the self-assembly of the collagen structures. By following the changes in the physical properties, we identified four stages, nucleation and growth of collagen precursors, formation of tropocollagen molecules, assembly of tropocollagens into microfibrils, and alignment of microfibrils to generate microribbons. Some emerging collagen structures never matured, and after an existence of several seconds, they disappeared into the solution. The elastic modulus of a microfibril (∼4 MPa) implied very small stiffness (∼3 × 10-6 N/m). Those values amplified the amplitude of the collagen thermal fluctuations on the mica plane, which facilitated microribbon build-up.

Practical Guide to Single-Protein AFM Nanomechanical Spectroscopy Mapping: Insights and Pitfalls As Unraveled by All-Atom MD Simulations on Immunoglobulin G.

Atomic force microscopy is an invaluable characterization tool in almost every biophysics laboratory. However, obtaining atomic/sub-nanometer resolution on single proteins has thus far remained elusive-a feat long achieved on hard substrates. In this regard, nanomechanical spectroscopy mapping may provide a viable approach to overcome this limitation. By complementing topography with mechanical properties measured locally, one may thus enhance spatial resolution at the single-protein level. In this work, we perform all-atom molecular dynamics simulations of the indentation process on a single immunoglobulin G (IgG) adsorbed on a graphene slab. Our simulations reveal three different stages as a function of strain: a noncontact regime-where the mechanical response is linked to the presence of the water environment- followed by an elastic response and a final plastic deformation regime. In the noncontact regime, we are able to identify hydrophobic/hydrophilic patches over the protein. This regime provides the most local mechanical information that allows one to discern different regions with similar height/topography and leads to the best spatial resolution. In the elastic regime, we conclude that the Young modulus is a well-defined property only within mechanically decoupled domains. This is caused by the fact that the elastic deformation is associated with a global reorganization of the domain. Differences in the mechanical response are large enough to clearly resolve domains within a single protein, such as the three subunits forming the IgG. Two events, unfolding or protein slipping, are observed in the plastic regime. Our simulations allow us to characterize these two processes and to provide a strategy to identify them in the force curves. Finally, we elaborate on possible challenges that could hamper the interpretation of such experiments/simulations and how to overcome them. All in all, our simulations provide a detailed picture of nanomechanical spectroscopy mapping on single proteins, showing its potential and the challenges that need to be overcome to unlock its full potential.

Atomic-scale mapping of hydrophobic layers on graphene and few-layer MoS2 and WSe2 in water.

The structure and the role of the interfacial water in mediating the interactions of extended hydrophobic surfaces are not well understood. Two-dimensional materials provide a variety of large and atomically flat hydrophobic surfaces to facilitate our understanding of hydrophobic interactions. The angstrom resolution capabilities of three-dimensional AFM are exploited to image the interfacial water organization on graphene, few-layer MoS2 and few-layer WSe2. Those interfaces are characterized by the existence of a 2 nm thick region above the solid surface where the liquid density oscillates. The distances between adjacent layers for graphene, few-layer MoS2 and WSe2 are ~0.50 nm. This value is larger than the one predicted and measured for water density oscillations (~0.30 nm). The experiments indicate that on extended hydrophobic surfaces water molecules are expelled from the vicinity of the surface and replaced by several molecular-size hydrophobic layers.

Dynamics of breaking intermolecular bonds in high-speed force spectroscopy.

Atomic force microscope based single-molecule force spectroscopy provides a description of a variety of intermolecular interactions such as those occurring between receptor molecules and their ligands. Advances in force spectroscopy have enabled performing measurements at high-speeds and sub-microsecond resolutions. We report experiments performed on a biotin-avidin system that reveal that the measured force decreases with the loading rate at high rates. This result is at odds with the established Bell-Evans theory that predicts a monotonic increase of the rupture force with the loading rate. We demonstrate that inertial and hydrodynamic forces generated during the breaking of the bond dominate the measured force at high loading rates. We develop a correction factor to incorporate those effects into the Bell-Evans theory. The correction is necessary to obtain accurate values of the intermolecular forces at high speeds.

Unraveling capillary interaction and viscoelastic response in atomic force microscopy of hydrated collagen fibrils.

The mechanical properties of collagen fibrils depend on the amount and the distribution of water molecules within the fibrils. Here, we use atomic force microscopy (AFM) to study the effect of hydration on the viscoelastic properties of reconstituted type I collagen fibrils in air with controlled relative humidity. With the same AFM tip, we investigate the same area of a collagen fibril with two different force spectroscopy methods: force-distance (FD) and amplitude-phase-distance (APD) measurements. This allows us to separate the contributions of the fibril's viscoelastic response and the capillary force to the tip-sample interaction. A water bridge forms between the tip apex and the surface, causing an attractive capillary force, which is the main contribution to the energy dissipated from the tip to the specimen in dynamic AFM. The force hysteresis in the FD measurements and the tip indentation of only 2 nm in the APD measurements show that the hydrated collagen fibril is a viscoelastic solid. The mechanical properties of the gap regions and the overlap regions in the fibril's D-band pattern differ only in the top 2 nm but not in the fibril's bulk. We attribute this to the reduced number of intermolecular crosslinks in the reconstituted collagen fibril. The presented methodology allows the mechanical surface properties of hydrated collagenous tissues and biomaterials to be studied with unprecedented detail on the nanometer scale.