| Abstract:This work describes a method that utilizes a microfluidic gradient generator to develop lateral lipid gradients in supported lipid bilayers (SLB). This methodology that we have developed provides freedom of choice with respect to the lipid composition as well as the shape of the lipid gradient. For instance, the steepness of the gradient, within certain limits of its functional behavior, can be chosen based upon the experimental objectives. In addition, the device has the ability to create a protein or bivalent cation gradient in the aqueous phase above the lipid bilayer and to elicit a gradient specific response in the SLB. To show the broad applicability of this microfluidic device, we demonstrate that we can create a phosphoinositide gradient on various length scales, ranging from 2mm to 50?m. We further show that a Ca(2+) gradient in the aqueous layer above a mixed phosphatidylcholine/phosphatidylinositol-4,5-bisphosphate or phosphatidylserine lipid bilayer can elicit gradient specific anionic lipid/Ca(2+) domain formation. The biomimetic platform can be combined with a Total Internal Reflection Fluorescence (TIRF) microscopy setup, which allows for the convenient observation of the time evolution of the gradient and the interaction of ligands with the lipid bilayer. The method provides unprecedented access to study the dynamics and mechanics of protein-lipid interactions on membranes with micron level gradients, mimicking plasma membranes in organisms such as Dictyostelium discodeum and neutrophils from mammalian cells.
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