Jason L. Poulos

Electrowetting on dielectric-based microfluidics for integrated lipid bilayer formation and measurement

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.

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.

Automatable lipid bilayer formation and ion channel measurement using sessile droplets.

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.

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 ∼500 μm 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 GΩ( for 12 days. Measurements of the ion channel gramicidin A, α-hemolysin, and alamethicin incorporated into these membranes showed the same conductance behavior as conventional membranes.

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.

Masking apertures enabling automation and solution exchange in sessile droplet lipid bilayers

Reconstitution of ion channels and transmembrane proteins in planar lipid bilayer membranes allow for their scientific study in highly controlled environments. Recent work with lipid bilayers formed from mechanically joined monolayers has shown their potential for wider technological application, including automation and parallelization. However, bilayer areas are highly sensitive to variations in mechanical position and the bilayers themselves cannot withstand significant perfusion of adjacent solutions. Toward this end, here we describe use of an aperture that masks the monolayer contact area, enabling formation of highly consistent bilayer areas and significantly reducing their variation with changes in relative position of the monolayers. Further, use of the aperture enables flow of solution adjacent to the bilayer without rupture or significant change in bilayer area. The device design is scalable and compatible with SBS standard instrumentation and automation technology, potentially enabling its use for rapid, parallel automated measurements of ion channels for large scale scientific studies and pharmaceutical screening.

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.

Automatable lipid bilayer formation for ion channel studies

Transmembrane proteins and ion channels are important drug targets and have been explored as single molecule sensors. For these proteins to function normally they must be integrated within lipid bilayers; however, the labor and skill required to create artificial lipid bilayers have the limited the possible applications utilizing these proteins. In order to reduce the complexity and cost of lipid bilayer formation and measurement, we have modified a previously published lipid bilayer formation technique using mechanically contacted monolayers so that the process is automatable, requiring minimal operator input. Measurement electronics are integrated with the fluid handling system, greatly reducing the time and operator feedback characteristically required of traditional bilayer experiments. To demonstrate the biological functionality of the resultant bilayers and the systems capabilities as a membrane platform, the ion channel gramicidin A was incorporated and measured with this system.