Microfluidic formation of lipid bilayer array for membrane transport analysis

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Microfluidic 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
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Abstract

• 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.

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Conceptual 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.

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System 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.

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Procedures
(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.
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Diffusion 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.
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Measurement 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.
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Conclusion

• 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.

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• Thanks for your attention!