Microfluidic device as a facile in vitro tool to generate and investigate lipid gradients.

This work describes a method that utilizes a microfluidic gradient generator to develop lateral lipid gradients in supported lipid bilayers (SLB). The new methodology provides freedom of choice with respect to the lipid composition of the SLB. In addition, the device has the ability to create a protein or bivalent cation gradient in the aqueous phase above the lipid bilayer which can elicit a gradient specific response in the SLB. To highlight these features we demonstrate that we can create a phosphoinositide gradient on various length scales, ranging from 2mm to 50µm. We further show that a Ca2+ gradient in the aqueous phase above the SLB causes anionic lipid clustering mirroring the cation gradient. We demonstrate this effect for mixed phosphatidylcholine/phosphatidylinositol-4,5-bisphosphate bilayers and fora mixed phosphatidylcholine/phosphatidylserine bilayers. 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 membrane gradients observed in organisms such as Dictyostelium discodeum and neutrophils.