Automated lipid bilayer and ion channel measurement platform.

Ion channels and transmembrane proteins play key roles in a wide range of physiological processes. Engineered ion channels have been explored as highly sensitive single molecule sensors. Scientific and sensing measurements of ion channel conductance often utilize artificial lipid bilayers, which have shortcomings limiting their application. We describe a fully automated lipid bilayer formation system that integrates the measurement electronics within the fluidic controls. Unattended operation of this system resulted in highly reproducible automatic bilayer formation and ion channel measurement over dozens of consecutive trials. The fully automated, closed-loop control algorithm enabled autonomous operation of the platform, a step toward applications of ion channel measurements for remote sensing and pharmacological studies requiring minimal operator involvement.

Automatable production of shippable bilayer chips by pin tool deposition for an ion channel measurement platform.

As a step towards an automated and operator-free ion channel measurement platform we have previously demonstrated a solution formulation for artificial lipid bilayers that enabled the indefinite storage and shipping of frozen bilayer precursors. In this work, the solutions were deposited by hand. Here, we have adapted pin tools to deposit the bilayer precursor solutions onto multi-element arrays, a popular method for microarray solution deposition. The pin tools have enabled the deposited volume to be applied highly repeatably and controllably, resulting in reduction of bilayer formation times to <1 h. The pin tools are also compatible with computerized motion control platforms, enabling automated and high throughput production. We discuss these results and the prospects of this technology to produce high density bilayer arrays for high throughput measurement of ion channels incorporated into artificial bilayers.

Ion channel and toxin measurement using a high throughput lipid membrane platform.

Measurements of ion channels are important for scientific, sensing and pharmaceutical applications. Reconstitution of ion channels into lipid vesicles and planar lipid bilayers for measurement at the single molecule level is a laborious and slow process incompatible with the high throughput methods and equipment used for sensing and drug discovery. A recently published method of lipid bilayer formation mechanically combines lipid monolayers self-assembled at the interfaces of aqueous and apolar phases. We have expanded on this method by vertically orienting these phases and using gravity as the driving force to combine the monolayers. As this method only requires fluid dispensation, it is trivially integrated with high throughput automated liquid-handling robotics. In a proof-of-concept demonstration, we created over 2200 lipid bilayers in 3h. We show single molecule measurements of technologically and physiologically relevant ion channels incorporated into lipid bilayers formed with this method.

Long-term storable and shippable lipid bilayer membrane platform.

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.

Black lipid membranes stabilized through substrate conjugation to a hydrogel.

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.