Toshinori Motegi

Enhanced Brownian Ratchet Molecular Separation Using a Self-Spreading Lipid Bilayer

A new approach is proposed for two-dimensional molecular separation based on the Brownian ratchet mechanism by use of a self-spreading lipid bilayer as both a molecular transport and separation medium. In addition to conventional diffusivity-dependence on the ratchet separation efficiency, the difference in the intermolecular interactions between the target molecules and the lipid bilayer is also incorporated as a new separation factor in the present self-spreading ratchet system. Spreading at the gap between two ratchet obstacles causes a local change in the lipid density at the gap. This effect produces an additional opportunity for a molecule to be deflected at the ratchet obstacle and thus causes an additional angle shift. This enables the separation of molecules with the same diffusivity but with different intermolecular interaction between the target molecule and surrounding lipid molecules. Here we demonstrate this aspect by using cholera toxin subunit B (CTB)-ganglioside GM1 (GM1) complexes with different configurations. The present results will unlock a new strategy for two-dimensional molecular manipulation with ultrasmall devices.

Effective Brownian Ratchet Separation by a Combination of Molecular Filtering and a Self-Spreading Lipid Bilayer System

A new molecular manipulation method in the self-spreading lipid bilayer membrane by combining Brownian ratchet and molecular filtering effects is reported. The newly designed ratchet obstacle was developed to effectively separate dye-lipid molecules. The self-spreading lipid bilayer acted as both a molecular transport system and a manipulation medium. By controlling the size and shape of ratchet obstacles, we achieved a significant increase in the separation angle for dye-lipid molecules compared to that with the previous ratchet obstacle. A clear difference was observed between the experimental results and the simple random walk simulation that takes into consideration only the geometrical effect of the ratchet obstacles. This difference was explained by considering an obstacle-dependent local decrease in molecular diffusivity near the obstacles, known as the molecular filtering effect at nanospace. Our experimental findings open up a novel controlling factor in the Brownian ratchet manipulation that allow the efficient separation of molecules in the lipid bilayer based on the combination of Brownian ratchet and molecular filtering effects.

Electroformation from patterned single-layered supported lipid bilayers for formation of giant vesicles with narrow size distribution

We describe a method of producing solvent-free giant vesicles (GVs) with a narrow size distribution from patterned supported lipid bilayers (SLBs). The SLBs were prepared on a patterned SiO2 surface by vesicle fusion, and the GVs were formed from the SLBs by electroformation. Fluorescence observation showed the formation of single-layered SLBs on circular patterns of SiO2 with a diameter of 20 µm, the area of which corresponded to that of a unilamellar vesicle of 10 µm diameter. The electroformation from the patterned SLBs produced the GVs with a median diameter of 8.53 µm and a coefficient of variation of 10.9%.

Fluidity evaluation of cell membrane model formed on graphene oxide with single particle tracking using quantum dot

The lipid bilayer is the fundamental structure of plasma membranes, and artificial lipid bilayer membranes are used as model systems of cell membranes. Recently we reported the formation of a supported lipid bilayer (SLB) on graphene oxide (GO) by the vesicle fusion method. In this study, we conjugated a quantum dot (Qdot) on the SLB surface as a fluorescence probe brighter than dye-labeled lipid molecules, to qualitatively evaluate the fluidity of the SLB on GO by the single particle tracking method. We obtained the diffusion coefficient of the Qdot-conjugated lipids in the SLB on GO. We also performed the Qdot conjugation on the SLB containing a lipid conjugated with polyethylene glycol, to prevent the nonspecific adsorption of Qdots. The difference in the diffusion coefficients between the SLBs on the GO and the bare SiO2 regions was evaluated from the trajectory of single Qdot-conjugated lipid diffusing between the two regions.

Substrate-Induced Structure and Molecular Dynamics in a Lipid Bilayer Membrane

The solid-substrate-dependent structure and dynamics of molecules in a supported lipid bilayer (SLB) were directly investigated via atomic force microscopy (AFM) and single particle tracking (SPT) measurements. The appearance of either vertical or horizontal heterogeneities in the SLB was found to be strongly dependent on the underlying substrates. SLB has been widely used as a biointerface with incorporated proteins and other biological materials. Both silica and mica are popular substrates for SLB. Using single-molecule dynamics, the fluidity of the upper and lower membrane leaflets was found to depend on the substrate, undergoing coupling and decoupling on the SiO2/Si and mica substrates, respectively. The anisotropic diffusion caused by the locally destabilized structure of the SLB at atomic steps appeared on the Al2O3(0001) substrate because of the strong van der Waals interaction between the SLB and the substrate. Our finding that the well-defined surfaces of mica and sapphire result in asymmetry and anisotropy in the plasma membrane is useful for the design of new plasma-membrane-mimetic systems. The application of well-defined supporting substrates for SLBs should have similar effects as cell membrane scaffolds, which regulate the dynamic structure of the membrane.

Fluidity Evaluation of Cell Membrane Model Formed on Graphene Oxide with Single Particle Tracking Using Qdot

Lipid bilayer is the fundamental structure of plasma membranes, and behaves as the reaction field for various membrane reactions. Recently we established the formation of supported lipid bilayers (SLBs) on graphene oxide (GO) with the vesicle fusion method for the development of a new method to measure the behavior of biomolecules in lipid bilayers using GO. In this study, we conjugated quantum dots (Qdots) to the SLB surface, and evaluated the fluidity of the SLB on GO with single particle observation. We found several diffusing Qdots on the SLB on GO, and obtained the diffusion coefficient from their trajectories.