Paul B. Davies

Sum Frequency Generation (SFG) Vibrational Spectroscopy of Planar Phosphatidylethanolamine Hybrid Bilayer Membranes under Water

Sum frequency generation (SFG) spectroscopy has been used to study the structure of phosphatidylethanolamine hybrid bilayer membranes (HBMs) under water at ambient temperatures. The HBMs were formed using a modified Langmuir-Schaefer technique and consisted of a layer of dipalmitoyl phosphatidylethanolamine (DPPE) physisorbed onto an octadecanethiol (ODT) self-assembled monolayer (SAM) at a series of surface pressures from 1 to 40 mN m(-1). The DPPE and ODT were selectively deuterated so that the contributions to the SFG spectra from the two layers could be determined separately. SFG spectra in both the C-H and C-D stretching regions confirmed that a monolayer of DPPE had been adsorbed to the ODT SAM and that there were gauche defects within the alkyl chains of the phospholipid. On adsorption of a layer of DPPE, methylene modes from the ODT SAM were detected, indicating that the phospholipid had partially disordered the alkanethiol monolayer. SFG spectra recorded in air indicated that removal of water from the surface of the HBM resulted in disruption of the DPPE layer and the formation of phospholipid bilayers.

Structure of Mixed Phosphatidylethanolamine and Cholesterol Monolayers in a Supported Hybrid Bilayer Membrane Studied by Sum Frequency Generation Vibrational Spectroscopy

The structure of hybrid bilayer membranes (HBMs) containing either a pure cholesterol or mixed cholesterol/dipalmitoylphosphatidylethanolamine (DPPE) proximal layer adsorbed onto an octadecanethiol (ODT) self-assembled monolayer (SAM) on a gold substrate have been investigated by sum frequency generation (SFG) spectroscopy. The HBMs were formed by the adsorption of either a pure cholesterol or mixed DPPE/cholesterol monolayer from the air/water interface of a Langmuir-Blodgett trough at surface pressures of 1, 20, or 40 mN·m(-1). SFG spectra were also recorded of HBMs where cholesterol was replaced by cholesterol-d(7), in which the terminal isopropyl group of the alkyl chain of cholesterol was isotopically labeled. In order to isolate the contribution to the SFG spectra from the cholesterol in the mixed cholesterol/phospholipid films, DPPE-d was used, in which the alkyl chains of the phospholipid were deuterated. The infrared spectra of solvent-cast cholesterol and cholesterol-d(7) films were recorded to aid with assignment of the SFG spectra of the HBMs. Features corresponding to methyl, methylene, and methine stretches of cholesterol were identified in the SFG spectra. Information on the polar orientation of SFG-active groups was obtained from the phases of the spectral features. The structure of the HBMs showed little dependence on the surface pressure at which they were formed. SFG spectra of HBMs with a mixed cholesterol/DPPE proximal layer were very similar to the spectra of HBMs with a pure cholesterol proximal layer, although the features in the spectra were more intense than anticipated for a film with half the number of cholesterol molecules, indicating that DPPE did have some effect on the orientation of cholesterol molecules in the film.

Interfacial structure of soft matter probed by SFG spectroscopy.

Sum frequency generation (SFG) vibrational spectroscopy, an interface-specific technique in contrast to, for example, attenuated total reflectance spectroscopy, which is only interface sensitive, has been employed to investigate the surface and interface structure of soft matter on a molecular scale. The experimental arrangement required to carry out SFG spectroscopy, with particular reference to soft matter, and the analytical methods developed to interpret the spectra are described. The elucidation of the interfacial structure of soft matter systems is an essential prerequisite in order to understand and eventually control the surface properties of these important functional materials.

Structural Changes in a Polyelectrolyte Multilayer Assembly Investigated by Reflection Absorption Infrared Spectroscopy and Sum Frequency Generation Spectroscopy

The structure of polyelectrolyte multilayer films adsorbed onto either a per-protonated or per-deuterated 11-mercaptoundecanoic acid (h-MUA/d-MUA) self assembled monolayer (SAM) on gold was investigated in air using two surface vibrational spectroscopy techniques, namely, reflection absorption infrared spectroscopy (RAIRS) and sum frequency generation (SFG) spectroscopy. Determination of film masses and dissipation values were made using a quartz crystal microbalance with dissipation monitoring (QCM-D). The films, containing alternating layers of the polyanion poly[1-[4-(3-carboxy-4-hydroxyphenylazo) benzenesulfonamido]-1,2-ethanediyl, sodium salt] (PAZO) and the polycation poly(ethylenimine) (PEI) built on the MUA SAM, were formed using the layer-by-layer electrostatic self-assembly method. The SFG spectrum of the SAM itself comprised strong methylene resonances, indicating the presence of gauche defects in the alkyl chains of the acid. The RAIRS spectrum of the SAM also contained strong methylene bands, indicating a degree of orientation of the methylene groups parallel to the surface normal. Changes in the SFG and RAIRS spectra when a PEI layer was adsorbed on the MUA monolayer showed that the expected electrostatic interaction between the polymer and the SAM, probably involving interpenetration of the PEI into the MUA monolayer, caused a straightening of the alkyl chains of the MUA and, consequently, a decrease in the number of gauche defects. When a layer of PAZO was subsequently deposited on the MUA/PEI film, further spectral changes occurred that can be explained by the formation of a complex PEI/PAZO interpenetrated layer. A per-deuterated MUA SAM was used to determine the relative contributions from the adsorbed polyelectrolytes and the MUA monolayer to the RAIRS and SFG spectra. Spectroscopic and adsorbed mass measurements combined showed that as further bilayers were constructed the interpenetration of PAZO into preadsorbed PEI layers was repeated, up to the formation of at least five PEI/PAZO bilayers.

The Structure of Lipid Bilayers Adsorbed on Activated Carboxy-Terminated Monolayers Investigated by Sum Frequency Generation Spectroscopy

The formation and structure of isotopically asymmetric supported bilayer membranes (SBMs) has been investigated using sum frequency generation (SFG) vibrational spectroscopy supplemented by reflection absorption infrared spectroscopy (RAIRS). The bilayers were composed of a proximal and distal leaflet of the phospholipid dipalmitoyl phosphatidylethanolamine (DPPE) supported on a gold surface. The proximal leaflet was chemically tethered to the gold via an 11-mercapto-undecanoic acid (MUA) self-assembled monolayer (SAM) that had been chemically modified to produce an activated succinimidyl ester headgroup using N-hydroxysuccinimide (NHS) and N-(3-dimethylaminopropyl)-N-ethylcarbodiimide (EDC). The activation of the MUA and the tethering of the DPPE were monitored and confirmed using SFG and RAIRS. The distal leaflet of the bilayer was added using either vesicle fusion (VF) or Langmuir-Blodgett (LB) deposition. To gain insight into the structure of each layer of the SBM perdeuterated DPPE (d-DPPE) and MUA (d-MUA) were used to create SBMs with a layer that was isotopically distinguishable from the rest. The polar orientation and conformational ordering of the lipids was determined using SFG. It was found that the tethering of the proximal lipid leaflet resulted in an increase in the conformational order of the MUA SAM. Furthermore, by careful analysis and comparison of spectra recorded in both the C-H (2800-3000 cm(-1)) and C-D (2000-2300 cm(-1)) stretching regions it was concluded that a better ordered and more biologically relevant lipid bilayer was formed when the distal leaflet was added using LB deposition. On the other hand the SFG spectra of the SBMs in which the distal leaflet was added by VF showed little evidence of conformational ordering on the time scale of minutes, suggesting the presence of an incomplete monolayer or of multilayer formation.

Sum Frequency Generation Vibrational Spectroscopy of Cholesterol in Hybrid Bilayer Membranes

The assignment of the vibrational spectrum of cholesterol is surprisingly incomplete for such a fundamental molecule. To improve our understanding, a new investigation of the spectra of cholesterol in the C-H stretching region has been undertaken using the surface specific technique of Sum Frequency Generation (SFG) vibrational spectroscopy and the complementary technique of Reflection Absorption Infrared Spectroscopy (RAIRS). They were used to record the spectra of monolayers of cholesterol in hybrid bilayer membranes (HBMs). In addition to cholesterol, spectra were recorded of HBMs comprising monolayers of the partially deuterated cholesterol isotopologues d6-cholesterol and d7-cholesterol, and the cholesterol analogues cholestanol and androstanol to aid assignment of the spectra. Monolayers of each of the five molecules were used to form the distal leaflet of HBMs with the proximal leaflet consisting of a monolayer of deuterated mercaptoundecanoic acid (d-MUA) self-assembled on a gold substrate. Although cholesterol has five methyl groups and eleven methylene groups, by using molecules in which certain groups were either deuterated or entirely absent, it was possible to assign vibrational bands to specific sets of methyl or methylene groups either in the alkyl chain or sterol ring system of the molecule. Analysis of the spectra showed that the alkyl chains of cholesterol are orientated away from the substrate, which is opposite to their orientation in HBMs when the proximal leaflet is a hydrophobic self-assembled monolayer of octadecane thiol (ODT) adsorbed on gold. Additionally it was shown that in the d-MUA HBM, the α-face of the cholesterol ring is inclined toward the layer of air above the film, and the β-face is inclined toward the gold substrate.

Effect of multiple group orientations on sum frequency generation spectra

Sum Frequency Generation (SFG) spectra of monolayer films on metal surfaces have been simulated where the monolayer contains methyl groups in two different orientations. The two methyl group tilt angles were taken into account by including a second resonant term in the expression for the second-order non-linear susceptibility of the film, χR, and allowing the two resonant terms to vary independently with tilt angle. The metal surface gives rise to a constant non-resonant susceptibility, χNR. The phases of the SFG signals are determined by the interference between the two resonant susceptibilities and the non-resonant susceptibility of the metal surface. Whether the features corresponding to the methyl symmetric (r+) and asymmetric (r−) stretching modes appeared as peaks or dips in the simulated spectra showed a strong dependence on the two tilt angles, and a weaker dependence on R, the ratio of the two hyperpolarisabilities β aca and β ccc. From the simulated spectra it was determined that there are combinations of the two methyl group tilt angles that result in the symmetric and asymmetric methyl resonances having different spectral profiles with one a peak and the other a dip. A general method has been developed from which boundary conditions for the two methyl group tilt angles can be formulated based on the experimentally determined resonant phases of the methyl symmetric and asymmetric stretching modes.

Orientation of cholesterol in hybrid bilayer membranes calculated from the phases of methyl resonances in sum frequency generation spectra

The phases of Sum Frequency Generation (SFG) vibrational resonances recorded from thin films on metal surfaces provide information on the orientation and tilt angles of the functional groups of molecules in the film. SFG spectra have been simulated for monolayer films in which the adsorbed molecule has an unequal number of methyl groups oriented in two different directions. The phases, on resonance, of the methyl symmetric (r(+)) and asymmetric (r(-)) resonances are determined as a function of the two methyl group tilt angles and the fraction of groups pointing in that particular direction. The results are first presented as two-dimensional projection plots for the r(+) and r(-) resonances and then combined to show the tilt angles of the methyl groups, and fraction of methyl groups in that orientation, for which both resonances are in phase or out of phase with one another. Mathematical expressions have been calculated to identify the precise boundary conditions for when the phases of the r(+) and r(-) resonances change. The results of these simulations are compared to the phases of the methyl resonances in experimental SFG spectra of d7-cholesterol in a hybrid bilayer membrane.

Structure of the Fundamental Lipopeptide Surfactin at the Air/Water Interface Investigated by Sum Frequency Generation Spectroscopy

The lipopeptide surfactin produced by certain strains of Bacillus subtilis is a powerful biosurfactant possessing potentially useful antimicrobial properties. In order to better understand its surface behavior, we have used surface sensitive sum frequency generation (SFG) vibrational spectroscopy in the C-H and C═O stretching regions to determine its structure at the air/water interface. Using surfactin with the leucine groups of the peptide ring perdeuterated, we have shown that a majority of the SFG signals arise from the 4 leucine residues. We find that surfactin forms a robust film, and that its structure is not affected by the number density at the interface or by pH variation of the subphase. The spectra show that the ring of the molecule lies in the plane of the surface rather than perpendicular to it, with the tail lying above this, also in the plane of the interface.

A structural and temporal study of the surfactants behenyltrimethylammonium methosulfate and behenyltrimethylammonium chloride adsorbed at air/water and air/glass interfaces using sum frequency generation spectroscopy

Molecular scale information about the structure of surfactants at interfaces underlies their application in consumer products. In this study the non-linear optical technique of Sum Frequency Generation (SFG) vibrational spectroscopy has been used to investigate the structure and temporal behaviour of two cationic surfactants used frequently in hair conditioners. SFG spectra of films of behenyltrimethylammonium methosulfate (BTMS) and behenyltrimethylammonium chloride (BTAC) were recorded at the air/water interface and on glass slides following Langmuir Blodgett (LB) deposition. The assignment of the BTMS and BTAC spectral features (resonances) to the CH stretching modes of the surfactants was consolidated by comparison with the SFG spectrum of deuterated cetyltrimethylammonium bromide (d-CTAB) and by recording spectra on D2O as well as on water. The CH resonances arise from the methylene and methyl groups of the tail and head-groups of the surfactants. A slow collapse mechanism was observed following film compression of both BTAC and BTMS. The change in molecular structure of the films undergoing this slow collapse was followed by recording sequential SFG spectra in the CH region, and by monitoring the SFG intensity at specific wavenumbers over time. Additionally, LB deposition onto glass was used to capture the state of the film during the slow collapse, and these SFG spectra showed close similarity to the corresponding spectra on water. Complementary Atomic Force Microscopy (AFM) was used to elucidate the layering of the compressed and relaxed films deposited onto mica by LB deposition.