Fabrication of a Nano-bio Device Using Giant Unilamellar Vesicles


Koji Sumitomo, Arianna McAllister, Yukihiro Tamba*, Youichi Shinozaki,
and Keiichi Torimitsu
Materials Science Laboratory, *Suzuka Nat. Coll. Tech.

To fabricate a nano-bio device that functions with membrane proteins, we are trying to form micron-scale wells covered with a lipid bilayer on a Si substrate [1]. The membrane proteins in the suspended membrane will be able to function as well as membrane proteins in vivo. In this study, we used giant unilamellar vesicles (GUVs) several tens of microns in diameter to form a lipid bilayer suspended over the wells.
GUVs have been studied as a simplified cell membrane model. Figure 1 shows a functional analysis of ion channels (gramicidin A) arranged into a GUV lipid membrane based on an analysis of changes in fluorescent intensity. The GUVsf internal environment includes the fluorescent probe (pyranine), which changes its emission intensity with pH. When the environment outside the GUVs is changed (pH is increased), the cations pass through the ion channels and cancel out the concentration gradient between the inside and outside of the GUVs. The increase in the pH of the GUVs with time is clearly shown.
Then, the GUVs were ruptured over the wells which were fabricated on the Si substrate using photolithography. The aperture of the wells has an overhang shape that was formed by selectively etching the Si substrate below the SiO2 layer. The wells were loaded with fluorescent probes and sealed with a lipid bilayer. Excess probes were rinsed out. Figure 2 shows that the fluorescent probes are trapped in the wells (b), although several holes around the patch are unfilled (a). The overhang shape improves the probability of the confinement of the fluorescent probes in the wells because the suspended lipid bilayer is prevented from falling into the wells. The fluorescence from the probes confined in the wells remains unchanged for one hour or more. This indicates that the probes remain stably in the wells without flowing out. When we changed the pH outside the wells, we could analyze the function of the ion channels in the suspended lipid bilayer, as in Fig. 1. We show that micron-scale wells for the functional analysis of membrane proteins are formed on the Si substrate.
The transport of ions through the ion channels will be analyzed by both the fluorescent intensity change and the ion current, which will be collected by an electrode embedded in the wells. This constitutes a major step toward the fabrication of nano-bio devices.

[1] Y. Shinozaki et al., Appl. Phys. Express 3 (2010) 027002.

Fig. 1. Functional analysis of ion channels on GUVs
(change of fluorescent intensity).
Fig. 2. Sealing of micron-scale wells on a
Si substrate with a lipid bilayer.