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 2 mm to 50 μm. 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.

这项工作描述了一种方法，该方法利用微流体梯度发生器在支持的脂质双层（SLB）中产生侧向脂质梯度。新的方法论为SLB的脂质组成提供了选择的自由。另外，该装置具有在脂质双层以上的水相中产生蛋白质或二价阳离子梯度的能力，这可以引起SLB中的梯度特异性反应。为了突出这些特征，我们证明了我们可以在2 mm至50μm的各种长度范围内创建磷酸肌醇梯度。我们进一步证明了Ca ²⁺SLB上方水相中的H 2+梯度引起阴离子脂质聚集，反映了阳离子梯度。我们证明了混合磷脂酰胆碱/磷脂酰肌醇-4,5-双磷酸酯双层和混合磷脂酰胆碱/磷脂酰丝氨酸双层的这种作用。仿生平台可以与全内反射荧光（TIRF）显微镜设置相结合，从而可以方便地观察梯度的时间演变以及配体与脂质双层的相互作用。该方法提供了前所未有的途径来研究具有微米水平梯度的膜上蛋白质-脂质相互作用的动力学和力学，从而模拟了在双歧杆菌和中性粒细胞等生物体中观察到的质膜梯度。