No abstract

We present a microfluidic platform for the formation and electrical measurement of lipid bilayer membranes. Using electrowetting on dielectric (EWOD), two or more aqueous droplets surrounded by a lipid-containing organic phase were manipulated into contact to form a lipid bilayer at their interface. Thin-film Ag/AgCl electrodes integrated into the device enabled electrical measurement of membrane formation and the incorporation of gramicidin channels of two bilayers in parallel.

The fragility and short lifetimes characteristic of conventionally formed lipid bilayer membranes has necessitated their preparation to be at the time and point of use. By using high freezing-point lipid-solvent mixtures, the process of lipid bilayer self-assembly may be reversibly arrested. In solid form, the bilayer precursor can be stored indefinitely and is sufficiently robust to withstand commercial shipping. Upon thawing, bilayer self-assembly resumes, resulting in a biologically functional membrane. Combination of this membrane precursor with an inexpensive chip results in a compact, practical, and disposable platform for ion channel measurements.

Artificial lipid bilayer membranes have been used to reconstitute ion channels for scientific and technological applications. Membrane formation has traditionally involved slow, labor intensive processes best suited to small scale laboratory experimentation. We have recently demonstrated a high throughput method of membrane formation using automated liquid-handling robotics. We describe here the integration of membrane formation and measurement with two methods compatible with automation and high throughput liquid-handling robotics. Both of these methods create artificial lipid bilayers by joining lipid monolayers self-assembled at the interface of aqueous and organic phases using sessile aqueous droplets in contact with a measurement electrode; one using a pin tool, commonly employed in high throughput fluid handling assays, and the other using a positive displacement pipette. Membranes formed with both methods were high quality and supported measurement of ion channels at the single molecule level. Full automation of bilayer production and measurement with the positive displacement pipette was demonstrated by integrating it with a motion control platform.

Recent research in stabilizing lipid bilayer membranes has been directed toward tethering the membrane to a solid surface or contacting the membrane with a solid support such as a gel. It is also known that the solvent annulus plays an important role in lipid bilayer stability. In this work, the authors set out to stabilize the solvent annulus. Glass substrates with approximately 500 mum apertures were functionalized with 3-methacryloxypropyltrimethoxysilane to allow cross-linking with a surrounding polyethyleneglycol dimethacrylate hydrogel. The hydrogel makes a conformal mold around both the lipid bilayer and the solvent reservoir. Since the hydrogel is covalently conjugated with the glass substrate via vinyl groups, the solvent annulus is prevented from leaving the aperture boundary. Measurements of a membrane created with this approach showed that it remained a stable bilayer with a resistance greater than 1 GOmega for 12 days. Measurements of the ion channel gramicidin A, alpha-hemolysin, and alamethicin incorporated into these membranes showed the same conductance behavior as conventional membranes.