It is well recognized that interfacial structure of biomolecules plays crucial role in controlling their functions. Thus, it is essential to obtain information of molecular structure. Vibrational spectroscopy, which has been well used in characterization of molecular structure, is one of the useful techniques to study the structure and functions of protein and other biomaterials. However, conventional vibrational spectroscopy suffers from sensitivity too poor to detect the signal from the monolayer. In this review, we present applications of two vibrational techniques with high surface sensitivities, namely, surface enhanced infrared absorption spectroscopy (SEIRAS) and sum frequency generation (SFG) spectroscopy, for the functional studies of biointerfaces.

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Since phospholipids are the basic molecules of biomembranes, giant uni-lamellar vesicles (GUVs) of phospholipids with 1-10μm in diameter have been extensively studied to understand the actual behavior of biomembranes as a model cell. Phospholipids, however, normally grow as multi-lamellar vesicles which are not suitable for constructing the model cells. It has been recently shown that the addition of sugar or salt promotes the formation of GUVs when they are mixed with phospholipid films before hydration. The mechanism of the GUV formation induced by the additives was, however, not clarified. In this study, neutron reflectometry, small-angle X-ray scattering, and phase-contrast microscopy experiments were performed, and it is confirmed that the osmotic pressure induced by the additives in a lipid/additive mixture film promotes the formation of GUVs.

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Mobility of drugs and biomembrane constituents is a key to elucidate the membrane transport mechanism in the cell. Lipid bilayer membrane is a dynamic structure where molecules are always fluctuating under physiological conditions. The mechanism of drug transport is related to the molecular dynamics in such soft, fluid membrane interface. To gain insight into molecular movements in membranes, we develop a noninvasive method to monitor dynamic properties of drugs and lipid components in membranes by applying multinuclear high-resolution solution NMR in combination with the pulsed-field-gradient (PFG) technique. We have quantified the diffusivity, the kinetics of membrane binding, and the bound fraction of the drug in situ by using large unilamellar vesicles of egg phosphatidylcholine as model cell membranes. The combination of 1D and PFG NMR serves to quantify the kinetics of membrane binding where the bound and the free components are unable to distinguish because of the rapid exchange on the NMR timescale. A small-sized 5-fluorouracil and fluorinated bisphenol A are used as a model drug.

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