Asymmetric response of interfacial water to applied electric fields.

Our understanding of the dielectric response of interfacial water, which underlies the solvation properties and reaction rates at aqueous interfaces, relies on the linear response approximation: an external electric field induces a linearly proportional polarization. This implies antisymmetry with respect to the sign of the field. Atomistic simulations have suggested, however, that the polarization of interfacial water may deviate considerably from the linear response. Here we present an experimental study addressing this issue. We measured vibrational sum-frequency generation spectra of heavy water (D2O) near a monolayer graphene electrode, to study its response to an external electric field under controlled electrochemical conditions. The spectra of the OD stretch show a pronounced asymmetry for positive versus negative electrode charge. At negative charge below 5 × 1012 electrons per square centimetre, a peak of the non-hydrogen-bonded OD groups pointing towards the graphene surface is observed at a frequency of 2,700 per centimetre. At neutral or positive electrode potentials, this 'free-OD' peak disappears abruptly, and the spectra display broad peaks of hydrogen-bonded OD species (at 2,300-2,650 per centimetre). Miller's rule1 connects the vibrational sum-frequency generation response to the dielectric constant. The observed deviation from the linear response for electric fields of about ±3 × 108 volts per metre calls into question the validity of treating interfacial water as a simple dielectric medium.

Insights into the phase behavior at interfaces using vibrational sum frequency generation spectroscopy.

Phase separation is ubiquitous at the interface between two distinct phases. Physical transformation during phase separation often plays a crucial role in many important mechanisms, such as lipid phase separation, which is fundamental for transport through biological membranes. Phase separation can be complex, involving changes in the physical state and the reorganization of molecular structures, influencing the behavior and function of materials and biological systems. Surface-sensitive vibrational sum frequency generation (VSFG) spectroscopy provides a powerful tool for investigating these interfacial processes. As a non-linear optical technique, VSFG spectroscopy is sensitive to changes in molecular orientation and interactions at interfaces, making it an ideal method for studying phase separation processes. Here, we review the molecular interaction mechanisms underlying phase separation. We also explore the application of VSFG spectroscopy in studying phase separation processes at different interfaces. In particular, we focus on oil-water interfaces, which are relevant in environmental and industrial contexts; polymer and lipid surfaces, important for materials science and biological membranes; and intrinsically disordered protein systems, which play key roles in cellular function and disease.

Imaging Heterogeneous 3D Dynamics of Individual Solutes in a Polyelectrolyte Brush.

Understanding molecular transport in polyelectrolyte brushes (PEBs) is crucial for applications such as separations, drug delivery, anti-fouling, and biosensors, where structural features of the polymer control intermolecular interactions. The complex structure and local heterogeneity of PEBs, while theoretically predicted, are not easily accessed with conventional experimental methods. In this work, we use 3D single-molecule tracking to understand transport behavior within a cationic poly(2-(N,N-dimethylamino)ethyl acrylate) (PDMAEA) brush using an anionic dye, Alexa Fluor 546, as the probe. The analysis is done by a parallelized, unbiased 3D tracking algorithm. Our results explicitly demonstrate that spatial heterogeneity within the brush manifests as heterogeneity of single-molecule displacements. Two distinct populations of probe motion are identified, with anticorrelated axial and lateral transport confinement, which we believe to correspond to intra- vs inter-chain probe motion.

The competing influence of surface roughness, hydrophobicity, and electrostatics on protein dynamics on a self-assembled monolayer.

Surface morphology, in addition to hydrophobic and electrostatic effects, can alter how proteins interact with solid surfaces. Understanding the heterogeneous dynamics of protein adsorption on surfaces with varying roughness is experimentally challenging. In this work, we use single-molecule fluorescence microscopy to study the adsorption of α-lactalbumin protein on the glass substrate covered with a self-assembled monolayer (SAM) with varying surface concentrations. Two distinct interaction mechanisms are observed: localized adsorption/desorption and continuous-time random walk (CTRW). We investigate the origin of these two populations by simultaneous single-molecule imaging of substrates with both bare glass and SAM-covered regions. SAM-covered areas of substrates are found to promote CTRW, whereas glass surfaces promote localized motion. Contact angle measurements and atomic force microscopy imaging show that increasing SAM concentration results in both increasing hydrophobicity and surface roughness. These properties lead to two opposing effects: increasing hydrophobicity promotes longer protein flights, but increasing surface roughness suppresses protein dynamics resulting in shorter residence times. Our studies suggest that controlling hydrophobicity and roughness, in addition to electrostatics, as independent parameters could provide a means to tune desirable or undesirable protein interactions with surfaces.

Single-Molecule Dynamics Reflect IgG Conformational Changes Associated with Ion-Exchange Chromatography.

Conformational changes of antibodies and other biologics can decrease the effectiveness of pharmaceutical separations. Hence, a detailed mechanistic picture of antibody-stationary phase interactions that occur during ion-exchange chromatography (IEX) can provide critical insights. This work examines antibody conformational changes and how they perturb antibody motion and affect ensemble elution profiles. We combine IEX, three-dimensional single-protein tracking, and circular dichroism spectroscopy to investigate conformational changes of a model antibody, immunoglobulin G (IgG), as it interacts with the stationary phase as a function of salt conditions. The results indicate that the absence of salt enhances electrostatic attraction between IgG and the stationary phase, promotes surface-induced unfolding, slows IgG motion, and decreases elution from the column. Our results reveal previously unreported details of antibody structural changes and their influence on macroscale elution profiles.

Untying the Gordian KNOT: Unbiased Single Particle Tracking Using Point Clouds and Adaptive Motion Analysis.

Achieving mechanistic understanding of transport in complex environments such as inside cells or at polymer interfaces is challenging. We need better ways to image transport in 3-D and better single particle tracking algorithms to determine transport that are not systemically biased toward any classical motion model. Here we present an unbiased single particle tracking algorithm: Knowing Nothing Outside Tracking (KNOT). KNOT uses point clouds provided by iterative deconvolution to educate individual particle localizations and link particle positions between frames to achieve 2-D and 3-D tracking. Information from prior point clouds fuels an independent adaptive motion model for each particle to avoid global models that could introduce biases. KNOT competes with or surpasses other 2-D methods from the 2012 particle tracking challenge while accurately tracking adsorption dynamics of proteins on polymer surfaces and early endosome transport in live cells in 3-D. We apply KNOT to study 3-D endosome transport to reveal new physical insight into locally directed and diffusive transport in live cells. Our analysis demonstrates better accuracy in classifying local motion and its direction compared to previous methods, revealing intricate intracellular transport heterogeneities.

Transforming Separation Science with Single-Molecule Methods.

Empirical optimization of the multiscale parameters underlying chromatographic and membrane separations leads to enormous resource waste and production costs. A bottom-up approach to understand the physical phenomena underlying challenges in separations is possible with single-molecule observations of solute-stationary phase interactions. We outline single-molecule fluorescence techniques that can identify key interactions under ambient conditions. Next, we describe how studying increasingly complex samples heightens the relevance of single-molecule results to industrial applications. Finally, we illustrate how separation methods that have not been studied at the single-molecule scale can be advanced, using chiral chromatography as an example case. We hope new research directions based on a molecular approach to separations will emerge based on the ideas, technologies, and open scientific questions presented in this Perspective.

Acoustic Vibrations of Al Nanocrystals: Size, Shape, and Crystallinity Revealed by Single-Particle Transient Extinction Spectroscopy.

Acoustic vibrations in plasmonic nanoparticles, monitored by an all-optical means, have attracted significant increasing interest because they provide unique insight into the mechanical properties of these metallic nanostructures. Al nanostructures are a recently emerging alternative to noble metal nanoparticles, because their broad wavelength tunability and high natural abundance make them ideal for many potential applications. Here, we investigate the acoustic vibrations of individual Al nanocrystals using a combination of electron microscopy and single-particle transient extinction spectroscopy, made possible with a low-pulse energy, high sensitivity, and probe-wavelength-tunable, single-particle transient extinction microscope. For chemically synthesized, faceted Al nanocrystals, the observed vibration frequency scales with the inverse particle diameter. In contrast, triangularly shaped Al nanocrystals support two distinct frequencies, corresponding to their in- and out-of-plane breathing modes. Unlike ensemble measurements, which measure average properties, measuring the damping time of the acoustic vibrations for individual particles enables us to investigate variations of the quality factor on the particle-to-particle level. Surprisingly, we find a large variation in quality factors even for nanocrystals of similar size and shape. This observed heterogeneity appears to result from substantially varying degrees of nanoparticle crystallinity even for chemically synthesized nanocrystals.

Generalized method to design phase masks for 3D super-resolution microscopy.

Point spread function (PSF) engineering by phase modulation is a novel approach to three-dimensional (3D) super-resolution microscopy, with different point spread functions being proposed for specific applications. It is often not easy to achieve the desired shape of engineered point spread functions because it is challenging to determine the correct phase mask. Additionally, a phase mask can either encode 3D space information or additional time information, but not both simultaneously. A robust algorithm for recovering a phase mask to generate arbitrary point spread functions is needed. In this work, a generalized phase mask design method is introduced by performing an optimization. A stochastic gradient descent algorithm and a Gauss-Newton algorithm are developed and compared for their ability to recover the phase masks for previously reported point spread functions. The new Gauss-Newton algorithm converges to a minimum at much higher speeds. This algorithm is used to design a novel stretching-lobe phase mask to encode temporal and 3D spatial information simultaneously. The stretching-lobe phase mask and other masks are fabricated in-house for proof-of-concept using multi-level light lithography and an optimized commercially sourced stretching-lobe phase mask (PM) is validated experimentally to encode 3D spatial and temporal information. The algorithms' generalizability is further demonstrated by generating a phase mask that comprises four different letters at different depths.

Re-orientation of water molecules in response to surface charge at surfactant interfaces.

We present a measurement of molecular orientation of water at charged surfactant aqueous interfaces as a function of surface charge density. The polarization dependent spectral line shapes of the water bend mode were measured by vibrational sum-frequency generation at the positively charged surfactant cetyltrimethylammonium bromide (CTAB)/water interface and negatively charged surfactant sodium dodecyl sulfate/water interface. Orientational analysis using the water bend mode as a vibrational probe, within the electric dipole approximation, reveals structural differences between these surfaces and quantifies how different hydrogen bonded species re-orient around the surfactant head groups as the surface charge density changes. As the concentration of the positively charged surfactant (CTAB) increases, the surface water molecules with free-OH groups reorient their hydrogen away from the bulk water and the C2v axis closer to the surface normal. This suggests that these free-OH molecules are in general located above the positively charged head groups of CTAB, and thus, the charge-dipole interaction pulls their oxygen "down" and pushes hydrogen "up." On the contrary, water molecules with two donor hydrogen bonds re-orient their hydrogen toward the bulk water, likely because most of these molecules are below the CTAB surfactant head groups.

Concentration Dependent Approach for Accurate Determination of Two-Photon Absorption Cross-Section of Fluorescent dye Molecule.

We have investigated the concentration dependent of two-photon induced fluorescence (TPIF) in methanolic solution of Rhodamine 6G and Rhodamine B dye using 120 fs laser pulses at 780 nm, 76 MHz repetition rate. TPIF study of these dyes was compared with their respective one photon fluorescence intensity. We have shown the effect of chopper on TPIF intensity from Rhodamine dyes, which have shown direct influence on the determined TPA Cross section of these dyes.

Variable Non-Gaussian Transport of Nanoplastic on Supported Lipid Bilayers in Saline Conditions.

Nanoplastic-lipid interaction is vital to understanding the nanoscale mechanism of plastic adsorption and aggregation on a lipid membrane surface. However, a single-particle mechanistic picture of the nanoplastic transport process on a lipid surface remains unclear. Here, we report a salt-dependent non-Gaussian transport mechanism of polystyrene particles on a supported 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) lipid bilayer surface. Particle stickiness on the POPC surface increases with salt concentration, where the particles stay longer at the surface and diffuse to shorter distances. Additionally, a non-Gaussian diffusion state dominates the transport process at high salt concentrations. Our current study provides insight into the transport mechanism of polystyrene (PS) particles on supported lipid membranes, which is essential to understanding fundamental questions regarding the adsorption mechanisms of nanoplastics on lipid surfaces.

Imaging Switchable Protein Interactions with an Active Porous Polymer Support.

Mechanistic details about how local physicochemistry of porous interfaces drives protein transport mechanisms are necessary to optimize biomaterial applications. Cross-linked hydrogels made of stimuli-responsive polymers have potential for active protein capture and release through tunable steric and chemical transformations. Simultaneous monitoring of dynamic changes in both protein transport and interfacial polymer structure is an experimental challenge. We use single-particle tracking (SPT) and fluorescence correlation spectroscopy Super-resolution Optical Fluctuation Imaging (fcsSOFI) to relate the switchable changes in size and structure of a pH-responsive hydrogel to the interfacial transport properties of a model protein, lysozyme. SPT analysis reveals the reversible switching of protein transport dynamics in and at the hydrogel polymer in response to pH changes. fcsSOFI allows us to relate tunable heterogeneity of the hydrogels and pores to reversible changes in the distribution of confined diffusion and adsorption/desorption. We find that physicochemical heterogeneity of the hydrogels dictates protein confinement and desorption dynamics, particularly at pH conditions in which the hydrogels are swollen.

Mechanistic Understanding of the Phosphorylation-Induced Conformational Rigidity at the AMPA Receptor C-terminal Domain.

Phosphorylation at the intracellular C-terminal domain (CTD) of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors induces conformational rigidity. Such intracellular alterations to the AMPA receptor influence its functional responses, which are involved in multiple synaptic processes and neuronal signaling. The structure of the CTD still remains unresolved, which poses challenges toward providing a mechanism for the process of phosphorylation and deciphering the role of each phosphorylation step in causing the resultant conformational behavior. Herein, we utilize smFRET spectroscopy to understand the mechanism of phosphorylation, with the help of strategic point mutations that mimic phosphorylation. Our results reveal that first, phosphorylation at three target sites (S818, S831, and T840) is necessary for the change in the secondary structure of the existing disordered native sequence. Also, the results suggest that the formation of the tertiary structure through electrostatic interaction involving one specific phosphorylation site (S831) stabilizes the structure and renders conformational rigidity.

Phosphorylation Induces Conformational Rigidity at the C-Terminal Domain of AMPA Receptors.

The intracellular C-terminal domain (CTD) of AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptor undergoes phosphorylation at specific locations during long-term potentiation. This modification enhances conductance through the AMPA receptor ion channel and thus potentially plays a crucial role in modulating receptor trafficking and signaling. However, because the CTD structure is largely unresolved, it is difficult to establish if phosphorylation induces conformational changes that might play a role in enhancing channel conductance. Herein, we utilize single-molecule Förster resonance energy transfer (smFRET) spectroscopy to probe the conformational changes of a section of the AMPA receptor CTD, under the conditions of point-mutated phosphomimicry. Multiple analysis algorithms fail to identify stable conformational states within the smFRET distributions, consistent with a lack of well-defined secondary structure. Instead, our results show that phosphomimicry induces conformational rigidity to the CTD, and such rigidity is electrostatically tunable.

Vibrational Sum Frequency Generation Spectroscopy Measurement of the Rotational Barrier of Methyl Groups on Methyl-Terminated Silicon(111) Surfaces.

The methyl-terminated Si(111) surface possesses a 3-fold in-plane symmetry, with the methyl groups oriented perpendicular to the substrate. The propeller-like rotation of the methyl groups is hindered at room temperature and proceeds via 120° jumps between three isoenergetic minima in registry with the crystalline Si substrate. We have used line-shape analysis of polarization-selected vibrational sum frequency generation spectroscopy to determine the rotational relaxation rate of the surface methyl groups and have measured the temperature dependence of the relaxation rate between 20 and 120 °C. By fitting the measured rate to an Arrhenius dependence, we extracted an activation energy (the rotational barrier) of 830 ± 360 cm-1 and an attempt frequency of (2.9 ± 4.2) × 1013 s-1 for the methyl rotation process. Comparison with the harmonic frequency of a methyl group in a 3-fold cosine potential suggests that the hindered rotation occurs via uncorrelated jumps of single methyl groups rather than concerted gear-like rotation.

On the Assignment of the Vibrational Spectrum of the Water Bend at the Air/Water Interface.

We previously reported the spectrum of the water bend vibrational mode (ν2) at the air/water interface measured using sum-frequency generation (SFG). Here, we present experimental evidence to aid the assignment of the ν2 spectral features to H-bonded classes of interfacial water, which is in general agreement with two recent independently published theoretical studies. The dispersive line shape shows an apparent frequency shift between SSP and PPP polarization combinations (SFG-visible-infrared). This is naturally explained as an interference effect between the negative (1630 cm-1) and positive (1662 cm-1) peaks corresponding to "free-OH" and "H-bonded" species, respectively, which have different orientations and thus different amplitudes in SSP and PPP spectra. A surfactant monolayer of sodium dodecyl sulfate (SDS) was used to suppress the free OH species at the surface, and the corresponding SFG spectral changes indicate that these water molecules with one of the hydrogens pointing up into the air phase contribute to the negative peak at 1630 cm-1.

Insight into Water Structure at the Surfactant Surfaces and in Microemulsion Confinement.

Interactions with surfactant molecules can significantly alter the structure of interfacial water. We present a comparative study of water-surfactant interactions using two different spectroscopic approaches: water at planar surfactant monolayers by sum frequency generation (SFG) spectroscopy and interfacial water confined in reverse micelles formed by the same surfactants using IR absorption spectroscopy. We report spectral features in the OH-stretching region (3200-3700 cm-1) that are observed in both IR and SFG spectra, albeit with different relative amplitudes, for ionic surfactant sodium 1,4-bis-2-ethylhexylsulfosuccinate (AOT) and nonionic surfactant polyoxyethylene(4)lauryl ether (Brij L-4) reverse micelles in hexane and the corresponding monolayers at the air/water interface. A prominent feature in the SFG spectra of the OH stretch at 3560 cm-1 is attributed to water molecules that have a weak donor hydrogen bond to the surfactant headgroup. The same feature is observed in the IR spectra of reverse micelles after deconvoluting the interfacial versus bulk spectral contributions. We performed an orientational analysis of these water molecules utilizing the polarization-dependent SFG spectra, which shows an average tilt angle of the OH stretch of surfactant-bound water molecules of ∼155° with respect to the surface normal.