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 Reporter : d Tsu-wei Huang

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 α-Hemolysin antibiotic pores.

Conceptual diagram the microchannels with microchambers  Each chamber is sealed with a lipid bilayer.  Transport of fluorescent molecules through nanopores ( formed when α-Hemolysin monomers become heptamerized) is measured as a change of intensity under a fluorescent microscope.

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 O 2 plasma. (b) Magnified view of microchannels and microchambers. The channel height is 7 μm.

Procedures (a) saturate PDMS with water, (b) infuse buffer containing membrane proteins (α-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.

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 α-Hemolysin nanopores.

Measurement and Analysis (a) Fluorescent intensity in the microchambers over time. Without α-Hemolysin, decrease is only due to the photobleaching. Rapid decrease is observed with α-Hemolysin. (b) Transport rate k calculated from (a) (df/dt=-kf), which is proportional to the number of α-Hemolysin incorporated.

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 α- Hemolysin membrane proteins.

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