By introducing phase separation between liquid-order (L o ) and liquid-crystalline (L α ) domains into the freestanding lipid bilayer on the micropatterned substrate, the advance of the nanobiodevice which works with the membrane proteins is expected. However, it is known that the L α phase is preferred at a freestanding lipid bilayer than the L o phase because of the difference of their flexibility. We propose to control L o /L α phase separation in the freestanding lipid bilayer by supplying the necessary amount of unsaturated lipid molecules by the vesicle fusion method. Unsaturated lipids supplied by vesicle fusion at the edge of the lipid patch were agglomerated into the freestanding lipid bilayer through lateral diffusion in the supported lipid bilayer and resulted in forming L α phase domains. Due to the restriction on the supply amount of unsaturated lipids, the L o /L α phase separation in the freestanding lipid bilayer remained for longer than 24 h.

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Lipid bilayers suspended over microwells on Si substrates are promising platforms for nanobiodevices that mimic cell membranes. Using the biotin-avidin interaction, we have succeeded in selectively arranging vesicles on the freestanding region of a lipid bilayer. When ternary lipid mixtures of saturated lipid, unsaturated lipid, and cholesterol are used, they separate into liquid-order (Lo) and liquid-crystalline (Lα) domains. A freestanding lipid bilayer prefers the Lα-phase over the Lo-phase because of the difference in their flexibility. In addition, the type of biotinylated lipid determines whether it is localized in the Lα-phase domain or the Lo-phase domain. As a result, the biotinylated unsaturated lipids localized in the Lα-phase domain aggregate in the freestanding lipid bilayer, and vesicles labeled with biotin selectively bind to the freestanding lipid bilayer by the biotin-avidin interaction. This technique helps to introduce biomolecules into the freestanding lipid bilayer of nanobiodevices via vesicles.

The effectiveness of protein and peptide pharmaceuticals depends essentially on their intrinsic pharmacokinetics. Small-sized pharmaceuticals in particular often suffer from short serum half-lives due to rapid renal clearance. To improve the pharmacokinetics by association with serum albumin (SA) in vivo , we generated an SA-binding tag of a helix–loop–helix (HLH) peptide to be linked with protein pharmaceuticals. For use in future preclinical studies, screening of yeast-displayed HLH peptide libraries against human SA (HSA) and mouse SA (MSA) was alternately repeated to give the SA-binding peptide AY-VE, which exhibited cross-binding activities to HSA and MSA with K D of 65 and 20 nM, respectively. As a proof of concept, we site-specifically conjugated peptide AY-VE with insulin to examine its bioactivity in vivo . In mouse bioassay monitoring the blood glucose level, the AY-VE conjugate was found to have a prolonged hypoglycemic effect for 12 h. The HLH peptide tag is a general platform for extending the bioactivity of therapeutic peptides or proteins.