Artificial cell membranes (Lipid Bilayer) and membrane proteins

> Phospholipid molecules spontaneously form bilayer lipid membranes by

   Hydrophobic and Hydrophilic Interaction in the water.

> Membrane proteins perform  various functions in the cell membranes.

> Some membrane protein transport substances, energy and information between

   outside and inside the cells.

> Membrane proteins perform as the transporter of ions and medicines, the receptor

   of odorant and light, and the perception of mechanical stress

MEMS(MicroElectroMechanicalSystem) and membrane proteins

 In our laboratory,

> We manufacture the platform for preparing artificial cell membranes by using a micro electro

　mechanical system (MEMS).

> System construction for measuring the signals of membrane protein reconstituted in lipid bilayer

　membranes.

> Application for biosensor, drug development and physicochemical study in controlled nano-space.

> Utilizing molecules, proteins, DNA/RNA, lipid, cell, MEMS, microfluidics techniques in our study.

Micro-sized processing techniques (Lithography,　Milling)

> Microfabrication by photolithography with plasma-etching and exposure‑masking.

> CAD/CAM micromilling for plastic fabrication.

> Manufacture of electrodes by metal deposition and  confirmation of processing accuracy

   by a measuring microscope.

Droplet contact method: Formation of artificial cell membranes

> Applying of oil/lipid solution and buffer solution into a chamber, two lipid monolayers

   are formed at the surface of aqueous droplets.

> Two droplet contact together and bilayer lipid membrane can be formed.

> Artificial cell membranes mimicking natural cell membranes are able to be formed by

   various types of lipid molecules.

A electrical single molecule detection by nanopore sensing

> Ions pass through a nanopore (≈1.4 nm in diameter)  reconstituted in membranes

   cause current increase.

> A translocating molecules through the pore  show the blocking current.

> DNA unzipping make the blocking time longer.

> Application: drug testing, food inspection, and diagnostic pathology.

The mimetic device of cellular information transfer mechanism

> DNA computing, which uses programmed DNA as logic elements, enables large-scale

   parallel computation which is difficult for existing computers.

> Although this calculation requires longer detection time because of DNA amplification

   by PCR and detection by fluorescence imaging, we implemented a reduction of detection

   time by electrical detection using nanopore sensing.

> Nanopores enable the detection and  translocation to next droplet at the single molecule level.

Electrical molecular computing using nanopores

> Micro RNA(miRNA) is non-coding RNA, and it shows overexpress in cancer.

> The pattern recognition is necessary because expression pattern of miRNAs differ with cancer types.

> This system can detect overexpress miRNAs in cancer specific by combination of DNA computing

   and nanopore measurement.

> Application to simple diagnosis is expected.

Characterization of various nanopores

> Nanopores used for sensing include various types of biological and solid nanopores.

> Characterization of these nanopores can find out optimal nanopore for sensing.

> By using nanopores with large diameter, it is possible to detect large molecules such as

   diagnostic marker protein with high sensitivity.

High-throughput measurement and preparation of droplets

> We achieved an artificial system generating two conductance states by embedding

   a synthetic metal-organic porous (MOP) into lipid bilayers.

> Two-pore channels which are expressed ubiquitously in animals and plants control

   the resting membrane potential by using dual channels.

> We demonstrated two distinct ion conductance states by embedding a single MOP

   molecule with the geometry of an Archimedean cuboctahedron.

> The triangular and square apertures in the cuboctahedron work independently as

   ion-transporting pathways.

High-throughput measurement and preparation of droplets

> Antimicrobial peptides kill cell bacteria by forming nanopore into lipid bilayer.

> Pore-forming activity of the antimicrobial peptide is measured by electrical measurement.

> Current signals are classified by those of shapes, and we are enable to consider the

   movement of peptides on membrane.

> This study contributes to clear the mechanism of antimicrobial peptides and to apply

   them as antimicrobial drugs

High-throughput measurement and preparation of droplets

> Molecular robots are composed of sensors, calculators, and actuators that are

   all implemented in liposomes or hydrogels.

> Synthetic ion channels(nanopores) are potential candidates for the sensor sections

   of molecular robots.

High-throughput measurement and preparation of droplets

> The droplet contact method (DCM) has　been reported to prepare stable planar bilayer

   lipid membranes (pBLMs).

> In this study, we applied a DCM strategy to pBLM formation using microfluidic techniques

   and attempted to form double-stacked pBLMs in micro-meter scale.

>First, microchannels with micro pillars were designed via hydrodynamic simulations to

   form a five-layered flow with aqueous and lipid/oil solutions.

>Then, pBLMs were successfully formed by controlling the pumping pressure of the

   solutions and allowing contact between the two lipid monolayers.