Crystallization from the melt is a critical process governing the properties of semi-crystalline polymeric materials. While structural analyses of melting and crystallization transitions in bulk polymers have been widely reported, in contrast, those in thin polymer films on solid supports have been underexplored. Herein, in situ Raman microscopy and self-modeling curve resolution (SMCR) analysis are applied to investigate the temperature-dependent structural changes in poly(ethylene oxide) (PEO) films during melting and crystallization phase transitions. By resolving complex overlapping sets of spectra, SMCR analysis reveals that the thermal transitions of 50 µm thick PEO films comprise two structural phases: an ordered crystalline phase and a disordered amorphous phase. The ordered structure of the crystalline PEO film entirely disappears as the polymer is heated; conversely, the disordered structure of the amorphous PEO film reverts to the ordered structure as the polymer is cooled. Broadening of the Raman bands was observed in PEO films above the melting temperature (67 °C), while sharpening of bands was observed below the crystallization temperature (45 °C). The temperatures at which these spectral changes occurred were in good agreement with differential scanning calorimetry (DSC) measurements, especially during the melting transition. The results illustrate that in situ Raman microscopy coupled with SMCR analysis is a powerful approach for unraveling complex structural changes in thin polymer films during melting and crystallization processes. Furthermore, we show that confocal Raman microscopy opens opportunities to apply the methodology to interrogate the structural features of PEO or other surface-supported polymer films as thin as 2 µm, a thickness regime beyond the reach of conventional thermal analysis techniques.

Modification of silica interfaces by covalent attachment of functional ligands is a primary means of controlling the interfacial chemistry of porous silicas used in separations, environmental cleanup, and biosensing. Recently, modification of hydrophobic, n-alkyl-silane-functionalized interfaces has been achieved through self-assembly of zwitterionic phospholipids or mixed-charged surfactants to form "hybrid bilayers", producing interfaces that mimic lipid-bilayer partitioning and provide shape-selective partitioning of aromatic hydrocarbons. Charged headgroups, however, introduce electrostatic interactions that strongly influence the retention of ionizable solutes and require careful control over pH and ionic strength in the solution phase. In this work, we propose modification of C18-functionalized chromatographic silica surfaces through self-assembly of long-chain primary alcohols to form uncharged hybrid-bilayer surfaces. Hybrid bilayers formed from alcohols ranging from C12OH to C22OH are investigated with in situ confocal-Raman microscopy, and the spectra indicate that they form highly ordered n-alkane structures, with order increasing as a function of alcohol chain length. Temperature-dependent Raman spectra of C12OH-C22OH hybrid bilayers were collected to investigate their melting transitions. Multivariate curve resolution of these spectra show broad, two-component melting transitions, indicating alcohol and C18 alkyl chains melt simultaneously. These results suggest an interdigitated interfacial structure, where the hydrocarbon chains of the adsorbed alcohol extend into the underlying C18 chains, ordering both layers. Interdigitation is confirmed by a temperature-dependent study of a deuterated C16-OH bilayer, where spectrally resolved Raman bands from deuterated and protiated hydrocarbons melt together. Finally, n-alkyl alcohol bilayers were tested for protein repellency, where no protein adsorption was observed when equilibrated with ∼1 mg/mL bovine serum albumin. Bilayers C16OH in chain length are shelf stable at refrigerated temperatures for months. These results demonstrate long-chain alcohol bilayers can be utilized to control the interfacial hydrocarbon structure of C18-modified silica and have potential for use in separations, biosensing, and anti-biofouling applications.

This report describes an investigation of the role of H2O and DMF (N,N-dimethylformamide) as solvents on the electrochemical oxidation of oxalate (C2O42–). In H2O/0.1 M Na2SO4, the 2e– oxidation of C2O42– yields two CO2 molecules via a classical ECE mechanism, resulting in a single oxidation wave at 1.2 V vs Ag/AgCl. In contrast, two distinct oxidation waves are observed in DMF/0.1 M TBAP at ~0.1 V and ~0.7 V vs Ag/AgCl. The second wave at more positive potentials in DMF was first assigned by Maran and co-workers to the oxidation of the adduct C2O42–•CO2 formed from generation of CO2 during the first wave. Electronic interaction of C2O42– and CO2 stabilizes C2O42–, making it more difficult to oxidize than free C2O42–. Herein, we present cyclic voltammetry (CV), finite difference (FD) simulations, Raman spectroscopy, and ab initio molecular dynamics (AIMD) simulations to determine the thermodynamics and kinetics of C2O42–•CO2 formation, as well as the adduct structure. FD simulations of the voltammetric data provide approximate thermodynamic and kinetic parameters for C2O42–•CO2 formation in DMF, as well as a new value for the diffusion coefficient of C2O42– in DMF that differs substantially from previously reported values. Raman spectroscopy directly demonstrates C2O42–•CO2 formation in DMF, while AIMD simulations predict that the solvent-dependent thermodynamics and mechanism for C2O42– oxidation can be explained by C2O42––solvent and C2O42––CO2 interactions. A by-product of this investigation is the prediction from AIMD simulations that the C2O42–•CO2 interaction in DMF destabilizes CO2. Consistent with this finding, cyclic voltammetry demonstrates that the thermodynamic potential for CO2 reduction in DMF at a Hg electrode decreases by ~0.3 V upon addition of C2O42– to the solution.

Surface immobilization of DNA for biosensing or separations applications requires covalent attachment chemistry that is efficient, reproducible, and stable. In this work, an approach to link thiol-functionalized DNA to thiol-modified silica surfaces using N,N'-1,4-phenylene-bismaleimide is optimized by developing an efficient, one-pot synthesis of the maleimide-conjugated DNA followed by its immediate reaction with thiolated porous silica particles. The methodology takes advantage of a Michael addition reaction that couples a phenyl-bismaleimide cross-linking reagent and thiol-modified DNA to form a monomeric DNA-maleimide conjugate. The 1:1 stoichiometry of this reaction must be carefully controlled to avoid excess thiol-DNA, which generates unreactive bismaleimide-linked DNA dimers, or excess bismaleimide, which competes with the DNA-maleimide conjugate for reaction with the thiolated silica surface. To achieve control over the reaction forming the DNA conjugate, we adapt a fluorescence assay for free-thiols using 7-diethylamino-3-(4-maleimidophenyl)-4-methyl-coumarin (CPM) to determine the concentration of thiol-modified DNA that emerges from its synthesis, disulfide labeling, reduction to a thiol, and purification. The fluorescence response of the CPM reagent was calibrated using reduced glutathione as a standard, which allowed determination of the concentrations of thiolated-DNA and control over the stoichiometry of its reaction with a bismaleimide linker. The maleimide-conjugated DNA product thus formed was then reacted with thiolated-silica in order to bind the DNA to the internal surfaces of porous silica, whose surface populations were determined in individual particles by confocal Raman microscopy. Self-modeling curve resolution of the Raman spectra of surface-bound molecules validated the efficiency of the bismaleimide:thiolated DNA reaction, which provided stoichiometric control over formation of the monomeric DNA-maleimide conjugate and its optimized reaction with thiolated-silica surfaces.

Simultaneous multipass resistive-pulse sensing and fluorescence imaging have been used to correlate the size and fluorescence intensity of individual E. coli lipid liposomes composed of E. coli polar lipid extract labeled with membrane-bound 3,3-dioctadecyloxacarbocyanine (DiO) fluorescent molecules. Here, a nanopipette serves as a waveguide to direct excitation light to the resistive-pulse sensing zone at the end of the nanopipette tip. Individual DiO-labeled liposomes (>50 nm radius) were multipassed back and forth through the orifices of glass nanopipettes 110-to-150 nm radius via potential switching to obtain sub-nanometer sizing precision, while recording the fluorescence intensity of the membrane-bound DiO molecules. Fluorescence was measured as a function of liposome radius and found to be approximately proportional to the total membrane surface area. The observed relationship between liposome size and fluorescence intensity suggests that multi-vesicle liposomes emit greater fluorescence compared to unilamellar liposomes, consistent with all lipid membranes of the multi-vesicle liposomes containing DiO. Fluorescent and non-fluorescent liposomes are readily distinguished from each other in the same solution using simultaneous multipass resistive-pulse sensing and fluorescence imaging. A fluorescence ‘dead zone’ of ~1 m thickness just outside of the nanopipette orifice was observed during resistive-pulse sensing, resulting in ‘on/off’ fluorescent behavior during liposome multipassing. Our results provide a path forward to simultaneously characterize the size of biologically relevant nanoparticles (e.g., extracellular vesicles) and the presence of fluorescently labeled surface proteins.

Despite the success of surface-enhanced Raman spectroscopy (SERS) for detecting DNA immobilized on plasmonic metal surfaces, its quantitative response is limited by the rapid falloff of enhancement with distance from the metal surface and variations in sensitivity that depend on orientation and proximity to plasmonic "hot spots". In this work, we assess an alternative approach for enhancing detection by immobilizing DNA on the interior surfaces of porous silica particles. These substrates provide over a 1000-fold greater surface area for detection compared to a planar support. The porous silica substrate is a purely dielectric material with randomly oriented internal surfaces, where scattering is independent of proximity and orientation of oligonucleotides relative to the silica surface. We characterize the quantitative response of Raman scattering from DNA in porous silica particles with sequences used in previous SERS investigations of DNA for comparison. The results show that Raman scattering of DNA in porous silica is independent of distance of nucleotides from the silica surface, allowing detection of longer DNA strands with constant sensitivity. The surface area enhancement within particles is reproducible (<4% particle-to-particle variation) owing to the uniform internal pore structure and surface chemistry of the silica support. DNA immobilization with a bis-thiosuccinimide linker provides a Raman-active internal standard for quantitative interpretation of Raman scattering results. Despite the high (30 mM) concentrations of immobilized DNA within porous silica particles, they can be used to measure nanomolar binding affinities of target molecules to DNA by equilibrating a very small number of particles with a sufficiently large volume of low-concentration solution of target molecules.

Dimethylferrocene-modified linear poly(ethylenimine) (DMFc-LPEI) has been widely studied as a redox-mediating polymer for biosensor and biofuel cell applications. An increasing number of studies have shown the potential for DMFc-LPEI to immobilize oxidoreductase enzymes and to mediate electron transfer at electrode supports, making DMFc-LPEI a promising high current density bioanode material. Herein, we introduce the immobilization of DMFc-LPEI on 3-glycidoxypropyltrimethoxysilane-modified glass substrates. The epoxy-terminated functional groups on the substrate are used to covalently attach the polymer chains to the substrate by a simple nucleophilic substitution reaction, while a crosslinker, ethylene glycol diglycidyl ether, stabilizes a network of polymer chains using the same chemistry. Confocal Raman spectroscopy reveals that the vibrational bands of immobilized linear poly(ethylenimine) (LPEI) broaden compared to those of free solid-phase LPEI, suggesting that immobilized polymer film exists in an amorphous phase. The Raman spectra of DMFc-LPEI showed prominent vibrations corresponding to CH3 wagging mode at 1037 cm-1 and NH || bending mode at 1451 cm-1 of dimethylferrocene (DMFc) and LPEI, respectively. In the future, this chemistry will be extended to in-situ confocal Raman microscopy investigations of DMFc-LPEI on optically-transparent indium tin oxide electrodes to unravel the structural and conformational changes associated with varying the redox state of the DMFc mediator.

The discovery of DNA aptamers that bind biomolecular targets has enabled significant innovations in biosensing. Aptamers form secondary structures that exhibit selective high-affinity interactions with their binding partners. The binding of its target by an aptamer is often accompanied by conformational changes, and sensing by aptamers often relies on these changes to provide readout signals from extrinsic labels to detect target association. Many biosensing applications involve aptamers immobilized to surfaces, but methods to characterize conformations of immobilized aptamers and their in situ response have been lacking. To address this challenge, we have developed a structurally informative Raman spectroscopy method to determine conformations of the 15-mer thrombin-binding aptamer (TBA) immobilized on porous silica surfaces. The TBA is of interest because its binding of α-thrombin depends on the aptamer forming an antiparallel G-quadruplex, which is thought to drive signal changes that allow thrombin-binding to be detected. However, specific metal cations also stabilize the G-quadruplex conformation of the aptamer, even in the absence of its protein target. To develop a deeper understanding of the conformational response of the TBA, we utilize Raman spectroscopy to quantify the effects of the metal cations, K+ (stabilizing) and Li+ (nonstabilizing), on G-quadruplex versus unfolded populations of the TBA. In K+ or Li+ solutions, we then detect the association of α-thrombin with the immobilized aptamer, which can be observed in Raman scattering from the bound protein. The results show that the association of α-thrombin in K+ solutions produces no detectable change in aptamer conformation, which is found in the G-quadruplex form both before and after binding its target. In Li+ solutions, however, where the TBA is unfolded prior to α-thrombin association, protein binding occurs with the formation of a G-quadruplex by the aptamer.

The immobilization of DNA to surfaces is required for numerous biosensing applications related to the capture of target DNA sequences, proteins, or small-molecule analytes from solution. For these applications to be successful, the chemistry of DNA immobilization should be efficient, reproducible, and stable and should allow the immobilized DNA to adopt a secondary structure required for association with its respective target molecule. To develop and characterize surface immobilization chemistry to meet this challenge, it is invaluable to have a quantitative, surface-sensitive method that can report the interfacial chemistry at each step, while also being capable of determining the structure, stability, and activity of the tethered DNA product. In this work, we develop a method to immobilize DNA to silica, glass, or other oxide surfaces by carrying out the reactions in porous silica particles. Due to the high specific surface area of porous silica, the local concentrations of surface-immobilized molecules within the particle are sufficiently high that interfacial chemistry can be monitored at each step of the process with confocal Raman microscopy, providing a unique capability to assess the molecular composition, structure, yield, and surface coverage of these reactions. We employ this methodology to investigate the steps for immobilizing thiolated-DNA to thiol-modified silica surfaces through sequential Michael addition reactions with the cross-linker 1,4-phenylene-bismaleimide. A key advantage of employing a phenyl-bismaleimide over a comparable alkyl coupling reagent is the efficient conversion of the initial phenyl-thiosuccinimide to a more stable succinamic acid thioether linkage. This transformation was confirmed by in situ Raman spectroscopy measurements, and the resulting succinamic acid thioether product exhibited greater than 95% retention of surface-immobilized DNA after 12 days at room temperature in aqueous buffer. Confocal Raman microscopy was also used to assess the conformational freedom of surface-immobilized DNA by comparing the structure of a 23-mer DNA hairpin sequence under duplex-forming and unfolding conditions. We find that the immobilized DNA hairpin can undergo reversible intramolecular duplex formation based on the changes in frequencies and intensities of the phosphate backbone and base-specific vibrational modes that are informative of the hybridization state of DNA.

A quantitative description of ionophore-mediated ion transport is important in understanding ionophore activity in biological systems and developing ionophore applications. Herein, we describe the direct measurement of the electrical current resulting from K+ transport mediated by individual valinomycin (val) ionophores. Step fluctuations in current measured across a 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) bilayer suspended over a ∼400 nm radius glass nanopore result from dynamic partitioning of val between the bilayer and torus region, effectively increasing or decreasing the total number of val present in the membrane. In our studies, approximately 30 val are present in the membrane on average with a val entering or leaving the bilayer approximately every 50 s, allowing measurement of changes in electrical current associated with individual val. The single-molecule val(K+) transport current at 0.1 V applied potential is (1.3 ± 0.6) × 10-15 A, consistent with estimates of the transport kinetics based on large val ensembles. This methodology for analyzing single ionophore transport is general and can be applied to other carrier-type ionophores.

Immobilization of DNA to surfaces offers a convenient means of screening the binding affinity and selectivity of potential small-molecule therapeutic candidates. Unfortunately, most surface-sensitive methods for detecting these binding interactions are not informative of the molecular structure, information that is valuable for understanding the non-covalent interactions that stabilize binding. In this work, we report a method to meet this challenge by employing confocal Raman microscopy to quantify the association of a minor-groove-binding antimicrobial peptide, netropsin, to duplex DNA hairpin sequences immobilized on the interior surfaces of porous silica particles. To assess binding selectivity, particles functionalized with different sequences of DNA were equilibrated with solutions of 100 nM netropsin, and selective association was detected based on the presence of netropsin Raman scattering in the particles. The selectivity study revealed that netropsin binds to sequences of duplex DNA having AT-rich recognition regions. To quantify binding affinities, these AT-rich DNA sequences were equilibrated with a range of netropsin solution concentrations (1 to 100 nM). Raman scattering intensities of netropsin versus solution concentration were well described by single-binding-site Langmuir isotherms with nanomolar dissociation constants, in agreement with previous isothermal calorimetry and surface plasmon resonance results. Target sequence binding was accompanied with changes in netropsin and DNA vibrational modes consistent with the hydrogen bonding between the amide groups of netropsin and adenine and thymine bases in the DNA minor groove. The binding of netropsin to a control sequence lacking the AT-rich recognition region exhibited an affinity nearly 4 orders of magnitude weaker than found for the target sequences. The Raman spectrum of netropsin interacting with this control sequence showed broad pyrrole and amide mode vibrations at frequencies similar to a free solution, revealing less constrained conformations compared with the specific binding interactions observed with AT-rich sequences.

Phospholipid bilayers formed at solid-liquid interfaces have garneredinterest as mimics of cell membranes to model association reactions of proteins withlipid bilayer-tethered ligands. Despite the importance of understanding how liganddensity in a lipid bilayer impacts the protein-ligand association response, relating theligand-modified lipid fraction to the absolute density of solution-accessible ligands in alipid bilayer remains a challenge in interfacial quantitative analysis. In this work,confocal Raman microscopy is employed to quantify the association of anti-biotin IgGwith a small fraction of biotinylated lipids dispersed in either gel-phase or liquid-crystalline supported lipid bilayers deposited on the interior surfaces of wide-pore silicasurfaces. We examine the question of whether inter-leaflet lipid translocationcontributes to the population of solution-accessible biotin ligands on the distal leaflet of a supported lipid bilayer by comparingtheir protein accumulation response with ligands dispersed in lipid monolayers on nitrile-derivatized silica surfaces. The binding ofthe antibody to biotin ligands dispersed in gel-phase bilayers exhibited an equivalent biotin coverage response as the accumulation ofIgG onto gel-phase monolayers, indicating that gel-phase bilayer symmetry was preserved. This result contrasts with the similar to 60%greater anti-biotin capture observed atfluid-phase bilayers compared tofluid-phase monolayers prepared at equivalent biotinfractions. This enhanced protein capture is attributed to biotin-capped lipids being transferred from the surface-associated proximalleaflet of the bilayer to the solution-exposed distal leaflet by the inter-leaflet exchange or lipidflip-flop, a facile process influid-phasesupported lipid bilayers. The results suggest caution in interpreting the results of quantitative studies of protein binding to lipid-tethered ligands dispersed influid-phase phospholipid bilayers.

Shape selectivity is important in reversed-phase liquid chromatographic separations, where stationary phases are capable of separating geometric isomers, thereby resolving solutes based on their three-dimensional structure or shape rather than other chemical differences. Numerous chromatographic studies have been carried out using n-alkyl-chain-modified columns to understand how molecular shape affects retention. For polycyclic aromatic hydrocarbons (PAHs), it was found that planar compounds were selectively retained over nonplanar structures of comparable molecular weight on surfaces with longer n-alkyl chains, higher chain-density, or at lower temperatures, where selectivity likely arises with greater ordering of the n-alkyl chains. A limitation of these studies, however, is the small range of chain ordering that can be achieved and lack of a direct measure of the n-alkyl-chain order of the stationary phases. In this work, we employ a C18 stationary phase modified with a monolayer of phospholipid as a means of significantly varying the n-alkyl chain order. These hybrid-supported lipid bilayers, which have previously been employed as membrane-like stationary phases for measuring lipophilicity, provide a unique approach to control n-alkyl chain ordering by varying the acyl chain length and degree of unsaturation of the phospholipid modifier. The degree of alkyl-chain order of the resulting modified surfaces is determined from the ratio of trans- versus gauche-conformers, measured in situ within individual porous particles by confocal Raman microscopy. This methodology was also used to assess the affinity of these surfaces for planar versus nonplanar PAH molecules. The retention selectivity for the planar versus nonplanar compounds, thus determined, was found to vary significantly and systematically with the degree of order of the acyl/alkyl chains in the hybrid-supported lipid bilayers. The investigation also demonstrates the utility of confocal Raman microscopy for interrogating the impact of solute partitioning on stationary-phase structure within porous chromatographic particles.

A wide range of important biological processes occur at phospholipid membranes including cell signaling, where a peptide or small molecule targets a membrane-localized receptor protein. In this work, we report the adaptation of confocal Raman microscopy to quantify populations of unlabeled glucagon-like peptide-1 (GLP-1), a membrane-active 30-residue incretin peptide, in supported phospholipid bilayers deposited on the interior surfaces of wide-pore porous silica particles. Quantification of lipid bilayer-associated peptide is achieved by measuring the Raman scattering intensity of the peptide relative to that of the supported lipid bilayer, which serves as an internal standard. The dependence of the bilayer-associated GLP-1 population on the solution concentration of GLP-1 produces an isotherm used to determine the equilibrium constant for peptide-bilayer association and the maximum peptide surface coverage. The maximum coverage of GLP-1 in the lipid bilayer was found to be only 1/5th of a full monolayer based on its hydrodynamic radius. The saturation coverage, therefore, is not limited by the size of GLP-1 but by the ability of the bilayer to accommodate the peptide at high concentrations within the bilayer. Raman spectra show that GLP-1 association with the supported bilayer is accompanied by structural changes consistent with the intercalation of the peptide into the bilayer, where the observed increase in acyl-chain order would increase the lipid density and provide free volume needed to accommodate the peptide. These results were compared with previous measurements of the association of fluorescently labeled GLP-1 with a planar-supported bilayer; the unlabeled peptide exhibits a 3-fold greater affinity for the lipid bilayer on the porous silica support, suggesting that the fluorescent label alters the GLP-1 lipid bilayer association.

Peptide nucleic acid (PNA) is a unique synthetic nucleic acid analog that has been adopted for use in many biological applications. These applications rely upon the robust Franklin-Watson-Crick base pairing provided by PNA, particularly at lower ionic strengths. However, our understanding of the relationship between the kinetics of PNA:DNA hybridization and ionic strength is incomplete. Here we measured the kinetics of association and dissociation of PNA with DNA across a range of ionic strengths and temperatures at single-molecule resolution using total internal reflection fluorescence imaging. Unlike DNA:DNA duplexes, PNA:DNA duplexes are more stable at lower ionic strength, and we demonstrate that this is due to a higher association rate. While the dissociation rate of PNA:DNA duplexes is largely insensitive to ionic strength, it is significantly lower than that of DNA:DNA duplexes having the same number and sequence of base pairing interactions. The temperature dependence of PNA:DNA kinetic rate constants indicate a significant enthalpy barrier to duplex dissociation, and to a lesser extent, duplex formation. This investigation into the kinetics of PNA:DNA hybridization provides a framework towards better understanding and design of PNA sequences for future applications.

Infrared and Raman spectroscopy techniques were applied to investigate the drying and aggregation behavior of Nafion ionomer particles dispersed in aqueous solution. Gravimetric measurements aided the identification of gel-phase development within a series of time-resolved spectra that tracked transformations of a dispersion sample during solvent evaporation. A spectral band characteristic of ionomer sidechain end group vibration provided a quantitative probe of the dispersion-to-gel change. For sets of attenuated total reflection Fourier transform infrared (ATR FT-IR) spectra, adherence to Beer's law was attributed to the relatively constant refractive index in the frequency region of hydrated -SO3 - group vibrations as fluorocarbon-rich ionomer regions aggregate in forming the structural framework of membranes and thin films. Although vibrational bands associated with ionomer backbone CF2 stretching vibrations were affected by distortion characteristic of wavelength-dependent refractive index change within a sample, the onset of band distortion signaled gel formation and coincided with ionomer mass % values just below the critical gelation point for Nafion aqueous dispersions. Similar temporal behavior was observed in confocal Raman microscopy experiments that monitored the formation of a thin ionomer film from an individual dispersion droplet. For the ATR FT-IR spectroscopy and confocal Raman microscopy techniques, intensity in the water H-O-H bending vibrational band dropped sharply at the ionomer critical gelation point and displayed a time dependence consistent with changes in water content derived from gravimetric measurements. The reported studies lay groundwork for examining the impact of dispersing solvents and above-ambient temperatures on fluorinated ionomer transformations that influence structural properties of dispersion-cast membranes and thin films.

Confocal Raman microscopy was applied to quantify redox species present within the diffusion layer adjacent to an electrode surface under potentiostatic control. A glass microscope coverslip with a thin indium tin oxide (ITO) coating served as both the working electrode and optical window for a microscope-stage mountable spectroelectrochemical cell. A high numerical aperture objective mounted in an inverted microscope frame just below the stage brought excitation radiation through the coverslip window and to a tight focus a few micrometers above the ITO film surface. Species diffusing into the confocal probe volume defined by the excitation beam focus and the collected light region were detected, identified and quantified based on their Raman scattering frequencies and intensities. In measurements that interrogated the interconversion of ferrocyanide and ferricyanide ions as a function of applied voltage, least-squares regression analysis of spectral datasets predicted the formal potential and relative surface concentrations of the ions in good agreement with the expected Nernstian response. Preliminary studies of methyl viologen reduction at an ITO film/Nafion membrane interface were conducted and show the possibility for estimation of mass transport coefficients of redox species within ionic polymer materials.

Characterization of DNA at solid/liquid interfaces remains a challenge because most surface-sensitive techniques are unable to provide quantitative insight into the base content, length, or structure. Surface-enhanced Raman scattering measurements of DNA hybridization on plasmonic-metal substrates have been used to overcome small Raman-scattering cross-sections; however, surface-enhanced Raman spectroscopy measurements are not generally quantitative due to the fall-off in the scattering signal with the decay of the electric field enhancement from the surface, which also limits the length of oligonucleotides that can be investigated. In this work, we introduce an experimental methodology in which confocal Raman microscopy is used to characterize hybridization reactions of ssDNA immobilized at the solid/liquid interface of porous silica particles. By focusing the femtoliter confocal probe volume within a single porous particle, signal enhancement arises from the ∼1500-times greater surface area detected compared to a planar substrate. Because the porous support is a purely dielectric material, the scattering signal is independent of the proximity of the oligonucleotide to the silica surface. With this technique, we characterize a 19-mer capture strand and determine its hybridization efficiency with 9-mer and 16-mer target sequences from the scattering of a structurally insensitive phosphate-stretching mode. Changes in polarizability and frequency of scattering from DNA bases were observed, which are consistent with Watson-Crick base pairing. Quantification of base content from their duplex scattering intensities allows us to discriminate between hybridization of two target strands of equivalent length but with different recognition sequences. A duplex having a single-nucleotide polymorphism could be distinguished from hybridization of a fully complementary strand based on differences in base content and duplex conformation.

Bisection tasks that require individuals to identify the midpoint of a line are often used to assess the presence of biases to spatial attention in both healthy and patient populations. These tasks have helped to uncover a phenomenon called pseudoneglect, a bias towards the left-side of space in healthy individuals. First identified in the tactile domain, pseudoneglect has been subsequently demonstrated in other sensory modalities such as vision. Despite this, the specific reliability of pseudoneglect within individuals across tasks and time has been investigated very little. In this study, we investigated the reliability of response bias within individuals across four separate testing sessions and during three line bisection tasks: landmark, line bisection and tactile rod bisection. Strong reliability was expected within individuals across task and session. Pseudoneglect was found when response bias was averaged across all tasks, for the entire sample. However, individual data showed biases to both left and right, with some participants showing no clear bias, demonstrating individual differences in bias. Significant, cross-session within-individual reliability was found for the landmark and tactile rod bisection tasks respectively, but no significant reliability was observed for the line bisection task. These results highlight the inconsistent nature of pseudoneglect within individuals, particularly across sensory modality. They also provide strong support for the use of the landmark task as the most reliable measure of pseudoneglect.

Phospholipid bilayers deposited on a variety of surfaces provide models for investigation of the lipid membrane structure and supports for biocompatible sensors. Hybrid-supported phospholipid bilayers (HSLBs) are stable membrane models for these investigations, typically prepared by self-assembly of a lipid monolayer over an n-alkane-modified surface. HSLBs have been prepared on nalkyl chain-modified silica and used for lipophilicity-based chromatographic separations. The structure of these hybrid bilayers differs from vesicle membranes where the lipid head group spacing is greater due to interdigitation of the lipid acyl chains with the underlying n-alkyl chains bound to the silica surface. This interdigitated structure exhibits a broader melting transition at a higher temperature due to strong interactions between the lipid acyl chains and the immobile n-alkyl chains bound to silica. In the present work, we seek to reduce the interactions between a lipid monolayer and its supporting substrate by self-assembly of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) on porous silica functionalized with nitrile-terminated surface ligands. The frequency of Raman scattering of the surface -C N stretching mode at the lipid-nitrile interface is consistent with an n-alkane-like environment and insensitive to lipid head group charge, indicating that the lipid acyl chains are in contact with the surface nitrile groups. The head group area of this lipid monolayer was determined from the within-particle phospholipid concentration and silica specific surface area and found to be 54 +/- 2 A(2), equivalent to the head group area of a DMPC vesicle bilayer. The structure of these nitrile-supported phospholipid monolayers was characterized below and above their melting transition by confocal Raman microscopy and found to be nearly identical to DMPC vesicle bilayers. Their narrow gel-to-fluid-phase melting transition is equivalent to dispersed DMPC vesicles, suggesting that the acyl chain structure on the nitrile support mimics the outer leaflet structure of a vesicle membrane.