Detecting phase transitions in phosphatidylcholine vesicles by Raman microscopy and self-modeling curve resolution.

The study of phospholipid phase transitions is important for understanding drug- and protein-membrane interactions as well as other phenomena such as trans-membrane diffusion and vesicle fusion. A temperature-controlled stage on a confocal Raman microscope has allowed phase transitions in optically trapped phospholipid vesicles to be monitored. Raman spectra were acquired and analyzed using self-modeling curve resolution, a multivariate statistical analysis technique. This method revealed the subtle spectral changes indicative of sub- and pretransitions and main transitions in vesicles composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). The Raman scattering results were compared to differential scanning calorimetry (DSC) experiments and found to be in good agreement. This method of observing lipid phase transition profiles requires little sample preparation and a minimal amount of lipid (

Resolution of intermediate adsorbate structures in the potential-dependent self-assembly of n-hexanethiolate on Silver by in situ surface-enhanced Raman spectroscopy.

Resolution of the reaction steps and the associated component Raman spectra during the formation or desorption of self-assembled monolayers is challenging because intermediate adsorbate populations are present at low concentrations and their spectral bands overlap. By collecting Raman spectra versus applied potential into a two-dimensional data set, one can utilize multivariate statistical techniques to resolve the component concentration profiles along with their corresponding Raman spectra. In situ surface-enhanced Raman spectroscopy (SERS) spectra were collected during the potential-dependent formation and desorption (-1.50 to -0.70V versus Ag/AgCl) of n-hexanethiolate monolayer at a polycrystalline Ag electrode. Resolution of the pure component spectra from these components was accomplished by using self-modeling curve resolution (SMCR), which does not require a physical model. For monolayer adsorption, the potential-dependent Raman spectra could be described by three significant eigenvectors; the eigenvectors could be rotated into a set of pure component spectra and concentration profiles using a linear least-squares step to find a common plane in the space of the eigenvectors representing the linear combination of the real-component responses. The convex hull surrounding the data in the plane and positive amplitude criteria were utilized to identify the coordinates of the pure component responses. The C-S stretching vibrations of the resolved spectra show that the initial adsorbate is a gauche conformer, which allows the hydrocarbon chain to lie on the metal surface; a second phase arises at higher coverage with trans C-S conformation, where the hydrocarbon chains are oriented off the surface plane, and a final complete monolayer is formed with a well-ordered, all-trans C-S configuration. In contrast, desorption studies showed only two surface phases, the initial well-ordered monolayer and the low-density phase dominated by gauche conformations. The results illustrate the utility of self-modeling curve resolution to unravel interfacial reaction mechanisms and intermediate structures from two-dimensional SERS data, without requiring prior knowledge of a physical model for the process.

C18-modified metal-colloid substrates for surface-enhanced Raman detection of trace-level polycyclic aromatic hydrocarbons in aqueous solution.

Metal colloids immobilized on a glass support substrate are modified with a self-assembled alkylsilane (C18) layer to promote adsorption of polycyclic aromatic hydrocarbons from aqueous solutions. Detection of these compounds from low concentration solutions is accomplished by using surface-enhanced Raman scattering (SERS). SERS spectra of pyrene adsorbed to C18-modified immobilized silver colloids are dominated by Raman bands that are not consistent with pyrene and indicate that pyrene undergoes a chemical reaction at the surface. The origins of this surface product are investigated, and it is determined that silver and oxygen are required to form the product, whose Raman spectrum is consistent with oxidation to a quinone. When a C18-modified gold-colloid substrate is used, Raman scattering consistent with unreacted pyrene is observed. The adsorption and detection of pyrene adsorbed from low (2 ppb) concentration aqueous solutions onto C18-modified gold-colloid substrates is reported; naphthalene and phenanthrene are detected at approximately 5 ppb. Adsorption kinetics are rapid (<5 min), and the concentration-dependent SERS response is consistent with a Langmuir isotherm.

Electric-field control of the tautomerization and metal ion binding reactivity of 8-hydroxyquinoline immobilized to an electrode surface.

Surface-enhanced Raman scattering (SERS) spectra of a metal-complexing ligand, immobilized to a silver electrode surface, exhibits significant structural changes upon application of modest potentials. A detailed spectroscopic investigation shows that the potential applied to the electrode surface governs the tautomerization equilibrium of the immobilized ligand, p-((8-hydroxyquinoline)azo)benzenethiol (SHQ). Potential-dependent SERS spectra reveal that SHQ exists predominantly in a keto-hydrazone tautomeric form at applied potentials that are negative of -300 mV (vs Ag/AgCl), while the enol-azo tautomer is strongly favored at potentials positive of this value. The observed switching of the tautomer population occurs within a narrow range of applied potentials, approximately 200 mV (Ag/AgCl). Electrical control over the tautomerization equilibrium of immobilized SHQ governs the reactivity of the ligand toward metal ion complexation, where the enol-azo tautomer exhibits much greater affinity for metal ion binding compared to its keto-hydrazone counterpart. Accordingly, the potential applied to the electrode can be used to influence metal ion binding of immobilized SHQ through control over the tautomerization equilibrium, to produce an electrically switchable surface for metal ion complexation. Large differences in the electric dipole moment of the two tautomers, estimated from density functional theory calculations, suggested a model where the potential dependence arises from the interaction of the ligand dipole with electric fields that exist at a polarized electrode surface. This model accurately predicts the relative tautomer populations versus applied potential, at interfacial electric fields that are consistent with previous measurements of the vibrational Stark effect at polarized interfaces. Potential applications of this technology to several areas of analytical chemistry are considered.

Measuring diffusion of molecules into individual polymer particles by confocal Raman microscopy.

Raman microscopy is a powerful method for providing spatially resolved, chemically selective information about the composition of materials. With confocal collection optics, the method is well suited to the analysis of small particles in contact with liquid solutions. In this work, the transport of an organic solvent component into small polystyrene particles is investigated. An inverted confocal Raman microscope is used to acquire spectra from individual 75-microm polystyrene particles in contact with acetonitrile/water mixtures. Monitoring the Raman scattering from the C[triple bond]N stretching mode of acetonitrile provides a measure of solvent uptake into the polymer material. The small collection volume defined by the confocal optics provides the micrometer spatial resolution needed to track solvent concentration at different locations within the particle with 30-s time resolution. The volume fraction of acetonitrile in water in the surrounding solution was varied in order to determine the concentration dependence of the diffusion kinetics. Modeling the transport of molecules into a particle was addressed by using finite element methods for the evaluation of the coupled space- and time-dependent differential equations that govern the molecular transport. The results indicate that the diffusion coefficient changes with the local solvent concentration in the polymer. At longer times, with the highest acetonitrile concentrations, an evolution of the solvent transport mechanism was observed, from a diffusive rate that depends on local concentration to a linear increase in concentration with time accompanied by measurable swelling of the particle volume.

Templating of multiple ligand metal ion complexation sites in 8-hydroxyquinoline-modified silica sol-gel materials investigated by in situ Raman spectroscopy.

Metal ion templating in a sol-gel synthesis is used to develop multiligand 8-hydroxyquinoline binding sites in porous silica structures. The acid-base equilibria and the metal ion binding equilibria and stoichiometry of these materials are investigated by in situ Raman spectroscopy. This technique is capable of resolving spectral responses of the free ligand and its acid-base forms along the monomeric and dimeric ligand complexes with Cu2+. The proton-transfer equilibrium constants and first ligand binding equilibrium constant to Cu2+ for the metal ion-templated silica are equivalent to surface-immobilized 8HQ on silica gel. The second ligand binding constant to Cu2+, however, is comparable to the first ligand binding constant, which differs from free-solution behavior, where an order of magnitude smaller value is expected. The free energy available for binding the second ligand within the templated material is comparable to the first ligand, probably due to the nearly optimal location of the second ligand for binding, based on the templating that is done during this synthesis. The metal ion concentration responses of sol-gels prepared with varying amounts of metal ion during the syntheses were also tested, and the results indicate control over the fraction of templated binding sites.

In situ raman spectroscopy studies of metal ion complexation by 8-hydroxyquinoline covalently bound to silica surfaces.

Raman spectroscopy is applied to an investigation of the interfacial chemistry of silica-immobilized 8-hydroxyquinoline (8HQ) for binding of metal ions over a wide range of solution conditions. Since the derivatized silica has a high specific binding capacity, the mass of silica equilibrated with solution needs to be small for studies of reactions with trace-level (microM) metal ions; otherwise, the solution volume required to reach equilibrium becomes excessive. To address this problem, a small-volume flow cell is designed for this work using a fiber-optic Raman probe inserted directly into the packed end of a microcolumn, allowing excitation and collection of Raman scattering from less than 10 mg of derivatized silica. This cell is attached to a flow system that allows control of solution conditions while the response of the 8HQ-silica material is acquired by continuous monitoring of Raman scattering from the sample. Raman spectra of the deprotonated, neutral, protonated, and copper-complexed forms of the ligand can be distinguished, allowing proton-transfer and metal ion binding reactions of the ligand to be investigated. To account for the effects of changing surface potential on these reactions, zeta-potential measurements are made on the 8HQ-silica particles under the same solution conditions that are employed in the Raman scattering measurements. The observed pH dependence of metal ion binding was corrected for the effect of surface potential using the Boltzmann equation, and the resulting equilibrium constant for binding of Cu2+ was independent of metal ion concentration over a 100-fold range from 30 microM to 5 mM.

Confocal Raman microscopy for monitoring chemical reactions on single optically trapped, solid-phase support particles.

Optical trapping of small structures is a powerful tool for the manipulation and investigation of colloidal and particulate materials. The tight focus excitation requirements of optical trapping are well suited to confocal Raman microscopy. In this work, an inverted confocal Raman microscope is developed for studies of chemical reactions on single, optically trapped particles and applied to reactions used in solid-phase peptide synthesis. Optical trapping and levitation allow a particle to be moved away from the coverslip and into solution, avoiding fluorescence interference from the coverslip. More importantly, diffusion of reagents into the particle is not inhibited by a surface, so that reaction conditions mimic those of particles dispersed in solution. Optical trapping and levitation also maintain optical alignment, since the particle is centered laterally along the optical axis and within the focal plane of the objective, where both optical forces and light collection are maximized. Hour-long observations of chemical reactions on individual, trapped silica particles are reported. Using two-dimensional least-squares analysis methods, the Raman spectra collected during the course of a reaction can be resolved into component contributions. The resolved spectra of the time-varying species can be observed, as they bind to or cleave from the particle surface.