Ryugo Tero

Lipid Bilayer Membrane with Atomic Step Structure: Supported Bilayer on a Step-and-Terrace TiO2(100) Surface

The formation of a supported planar lipid bilayer (SPLB) and its morphology on step-and-terrace rutile TiO 2(100) surfaces were investigated by fluorescence microscopy and atomic force microscopy. The TiO 2(100) surfaces consisting of atomic steps and flat terraces were formed on a rutile TiO 2 single-crystal wafer by a wet treatment and annealing under a flow of oxygen. An intact vesicular layer formed on the TiO 2(100) surface when the surface was incubated in a sonicated vesicle suspension under the condition that a full-coverage SPLB forms on SiO 2, as reported in previous studies. However, a full-coverage, continuous, fluid SPLB was obtained on the step-and-terrace TiO 2(100) depending on the lipid concentration, incubation time, and vesicle size. The SPLB on the TiO 2(100) also has step-and-terrace morphology following the substrate structure precisely even though the SPLB is in the fluid phase and an approximately 1-nm-thick water layer exists between the SPLB and the substrate. This membrane distortion on the atomic scale affects the phase-separation structure of a binary bilayer of micrometer order. The interaction energy calculated including DLVO and non-DLVO factors shows that a lipid membrane on the TiO 2(100) gains 20 times more energy than on SiO 2. This specifically strong attraction on TiO 2 makes the fluid SPLB precisely follow the substrate structure of angstrom order.

Substrate Effects on the Formation Process, Structure and Physicochemical Properties of Supported Lipid Bilayers

Supported lipid bilayers are artificial lipid bilayer membranes existing at the interface between solid substrates and aqueous solution. Surface structures and properties of the solid substrates affect the formation process, fluidity, two-dimensional structure and chemical activity of supported lipid bilayers, through the 1–2 nm thick water layer between the substrate and bilayer membrane. Even on SiO2/Si and mica surfaces, which are flat and biologically inert, and most widely used as the substrates for the supported lipid bilayers, cause differences in the structure and properties of the supported membranes. In this review, I summarize several examples of the effects of substrate structures and properties on an atomic and nanometer scales on the solid-supported lipid bilayers, including our recent reports.

Polymerized lipid bilayers on a solid substrate: morphologies and obstruction of lateral diffusion.

Substrate supported planar lipid bilayers (SPBs) are versatile models of the biological membrane in biophysical studies and biomedical applications. We previously developed a methodology for generating SPBs composed of polymeric and fluid phospholipid bilayers by using a photopolymerizable diacetylene phospholipid (DiynePC). Polymeric bilayers could be generated with micropatterns by conventional photolithography, and the degree of polymerization could be controlled by modulating UV irradiation doses. After removing nonreacted monomers, fluid lipid membranes could be integrated with polymeric bilayers. Herein, we report on a quantitative study of the morphology of polymeric bilayer domains and their obstruction toward lateral diffusion of membrane-associated molecules. Atomic force microscopy (AFM) observations revealed that polymerized DiynePC bilayers were formed as nanometer-sized domains. The ratio of polymeric and fluid bilayers could be modulated quantitatively by changing the UV irradiation dose for photopolymerization. Lateral diffusion coefficients of lipid molecules in fluid bilayers were measured by fluorescence recovery after photobleaching (FRAP) and correlated with the amount of polymeric bilayer domains on the substrate. Controlled domain structures, lipid compositions, and lateral mobility in the model membranes should allow us to fabricate model membranes that mimic complex features of biological membranes with well-defined structures and physicochemical properties.

Supported phospholipid bilayer formation on hydrophilicity-controlled silicon dioxide surfaces

We investigated the influence of surface hydroxyl groups (-OHs) on the supported planar phospholipid bilayer (SPB) formation and characteristics. We prepared SiO2 surfaces with different hydrophilicity degree by annealing the SiO2 layer on Si(100) formed by wet chemical treatments. The hydrophilicity reduced with irreversible thermal desorption of -OHs. We formed SPB of dimyristoylphosphatidylcholine on the SiO2 surfaces by incubation at a 100-nm-filtered vesicle suspension. The formation rate was faster on less hydrophilic surfaces. We proposed that a stable hydrogen-bonded water layer on the SiO2 surface worked as a barrier to prevent vesicle adhesion on the surface. Theoretical calculation indicates that water molecules on vicinal surface -OHs take a stable surface-unique geometry, which disappears on an isolated -OH. The surface -OH density, however, affected little the fluidity of once formed SPBs, which was measured by the fluorescence recovery after the photobleaching method. We also describe the area-selective SPB deposition using surface patterning by the focused ion beam.

Surface-induced phase separation of a sphingomyelin/cholesterol/ganglioside GM1-planar bilayer on mica surfaces and microdomain molecular conformation that accelerates Aβ oligomerization

Ganglioside GM1 mediates the amyloid beta (Abeta) aggregation that is the hallmark of Alzheimers disease (AD). To investigate how ganglioside-containing lipid bilayers interact with Abeta, we examined the interaction between Abeta40 and supported planar lipid bilayers (SPBs) on mica and SiO(2) substrates by using atomic force microscopy, fluorescence microscopy, and molecular dynamics computer simulations. These SPBs contained several compositions of sphingomyelin, cholesterol, and GM1 and were treated at physiological salt concentrations. Surprisingly high-speed Abeta aggregation of fibril formations occurred at all GM1 concentrations examined on the mica surface, but on the SiO(2) surface, only globular agglomerates formed and they formed slowly. At a GM1 concentration of 20mol%, unique triangular regions formed on the mica surface and the rapidly formed Abeta aggregations were observed only outside these regions. We have found that some unique surface-induced phase separations are induced by the GM1 clustering effects and the strong interactions between the GM1 head group and the water layer adsorbed in the ditrigonal cavities on the mica surface. The speed of Abeta40 aggregation and the shape of the agglomerates depend on the molecular conformation of GM1, which varies depending on the substrate materials. We identified the conformation that significantly accelerates Abeta40 aggregation, and we think that the detailed knowledge about the GM1 molecular conformation obtained in this work will be useful to those investigating Abeta-GM1 interactions.

Construction and structural analysis of tethered lipid bilayer containing photosynthetic antenna proteins for functional analysis.

The construction and structural analysis of a tethered planar lipid bilayer containing bacterial photosynthetic membrane proteins, light-harvesting complex 2 (LH2), and light-harvesting core complex (LH1-RC) is described and establishes this system as an experimental platform for their functional analysis. The planar lipid bilayer containing LH2 and/or LH1-RC complexes was successfully formed on an avidin-immobilized coverglass via an avidin-biotin linkage. Atomic force microscopy (AFM) showed that a smooth continuous membrane was formed there. Lateral diffusion of these membrane proteins, observed by a fluorescence recovery after photobleaching (FRAP), is discussed in terms of the membrane architecture. Energy transfer from LH2 to LH1-RC within the tethered membrane was observed by steady-state fluorescence spectroscopy, indicating that the tethered membrane can mimic the natural situation.

Anomalous Diffusion in Supported Lipid Bilayers Induced by Oxide Surface Nanostructures

Hierarchic structure and anomalous diffusion on submicrometer scale were introduced into an artificial cell membrane, and the spatiotemporal dependence of lipid diffusion was visualized on nanostructured oxide surfaces. We observed the lipid diffusion in supported lipid bilayers (SLBs) on step-and-terrace TiO(2)(100) and amorphous SiO(2)/Si surfaces by single molecule tracking (SMT) method. The SMT at the time resolution of 500 μs to 30 ms achieved observation of the lipid diffusion over the spatial and temporal ranges of 100 nm/millisecond to 1 μm/second. The temporal dependence of the diffusion coefficient in the SLB on TiO(2)(100) showed that the crossover from anomalous diffusion to random diffusion occurred around 10 ms. The surface fine architecture on substrates will be applicable to induce hierarchic structures on the order of 100 nm or less, which correspond to the microcompartment size in vivo.

Oxygen adsorption states on Mo(112) surface studied by HREELS

Oxygen adsorption on a Mo(1 1 2) has been studied using HREELS, LEED and TPD. Oxygen molecules dissociatively adsorb to occupy quasi-threefold hollow site, long bridge site and atop site on a clean Mo(1 1 2) at 100 K. After annealing this surface to 300 K, a loss peak corresponding to the oxygen atoms at atop sites disappears, changing the adsorption sites into quasi-threefold and long bridge sites. Further annealing to 600 K resulted in the change of the location of oxygen atoms from long bridge site to quasi-threefold hollow site, where the surface shows p(1×2) LEED subspots, indicating that the oxygen atoms in the Mo(1 1 2)–p(1×2)-O surface occupy quasi-threefold hollow sites. Oxygen adsorption on the Mo(1 1 2)–p(1×2)-O surface was also investigated and it was found that oxygen molecules associatively adsorb on the surface to give bridged peroxo species, which dissociates at 200 K to yield oxygen atoms at atop sites. The atop oxygen atoms remain on the surface after annealing to higher temperatures in contrast to the adsorption on clean Mo(1 1 2). Further annealing to 800 K resulted in a disordered surface associated with a molybdenum oxide (MoxOy) layer.

Plasma irradiation of artificial cell membrane system at solid-liquid interface

We provide direct evidence of plasma-induced pore formation in a cell membrane model system. We irradiated plasma on the basis of the dielectric barrier discharge onto a supported lipid bilayer (SLB). Observation with a fluorescence microscope and atomic force microscope revealed the formation of pores on the order of 10 nm–1 µm in size. Capturing these micropores in a fluid lipid membrane is a significant advantage of the SLB system, and quantitative analysis of the pores was performed. Stimulation with equilibrium chemicals (HNO3 and H2O2) indicated that other transient active species play critical roles during the poration in the SLB.

Microorganism mediated synthesis of reduced graphene oxide films

The wide-ranging industrial application of graphene and related compounds has led researchers to devise methods for the synthesis of high quality graphene. We recently reported on the chemical synthesis, patterning, and doping of graphene films by the chemical exfoliation of graphite into graphene oxide (GO) with subsequent chemical reduction into graphene films [1, 2]. Here, we describe a hybrid approach for the synthesis of reduced graphene sheets, where chemically derived GO was reduced by microorganisms extracted from a riverside near the University. Our procedure enabled the production of ~100 μm sized reduced graphene sheets, which showed excellent Raman spectra associated with high quality reduced graphene. We give a detailed account of the relationship between the type of microorganisms and the properties of the resulting reduced graphene.