Title: Microfluidic formation of lipid bilayer array for membrane transport analysis
1Microfluidic formation of lipid bilayer array for
membrane transport analysis
- Sadao Ota, Wei-Heong Tan, Hiroaki Suzuki, and
Shoji Takeuchi - Center for International Research on
Micromechatoronics, Institute of Industrial
Science, The University of Tokyo, JAPAN - PRESTO, JST
MEMS 2008.
Reporter d9511814 Tsu-wei Huang
2Abstract
- A highly parallel and reproducible method for
reconstituting an array of lipid bilayers to
analyze membrane transport. - Infuse buffer/lipid/buffer solutions sequentially
into a microchannel with numerous microchambers
in its walls and seal each chamber by a lipid
bilayer containing membrane proteins. - Successfully perform quantitative measurement of
the transport flux of fluorescent molecules
(Calcein) through a-Hemolysin antibiotic pores.
3Conceptual diagram the microchannels with
microchambers
- Each chamber is sealed with a lipid bilayer.
- Transport of fluorescent molecules through
nanopores ( formed when a-Hemolysin monomers
become heptamerized) is measured as a change of
intensity under a fluorescent microscope.
4System and Device Design
- (a) PDMS device and the infusion system.
- Different fluids loaded into the same tube are
sequentially infused into the microchannel using
a syringe pump. - This PDMS device consists of the fabricated PDMS
and a coverglass, which were bonded permanently
after being applied O2 plasma. - (b) Magnified view of microchannels and
microchambers. - The channel height is 7 µm.
5Procedures
(a) saturate PDMS with water, (b) infuse buffer
containing membrane proteins (a-Hemolysin) and
fluorescent dye (Calcein). (c) infuse hexadecane
containing dissolved phosphatidylcholine (PC)
lipids (d) finally flush the channels with
buffer nothing dissolves. (e)(f) Close-up
illustration and image of microchambers. A thin
solvent layer covering the microchamber thins
down by the buffer flow, and lipids are
reconstructed into a planar bilayer membrane.
(g) Close up image of a formed lipid bilayer in
a microchamber.
6Diffusion Phenomenon through lipid bi-layers
Time-lapsed images showing the rapid decrease in
fluorescent intensity in the microchambers due to
the diffusion of Calcein to the microchannel
through a-Hemolysin nanopores.
7Measurement and Analysis
- (a) Fluorescent intensity in the microchambers
over time. -
- Without a-Hemolysin, decrease is only due to the
photobleaching. - Rapid decrease is observed with a-Hemolysin.
(b) Transport rate k calculated from (a)
(df/dt-kf), which is proportional to the number
of a-Hemolysin incorporated.
8Conclusion
- The method to form artificial lipid bilayer array
inside a microfluidic device. - This method is simple and inexpensive
- The high-density array facilitates quantitative
measurements on transport phenomena through
membrane proteins. - Be able to exchange buffer in microchannels
without breaking the membranes or losing the
functionality of a-Hemolysin membrane proteins.
9- Thanks for your attention!