This paper presents a plug-based microfluidic system to dispense nanoliter-volume plugs of Lipidic Cubic Phase (LCP) material and subsequently merge the LCP plugs with aqueous plugs. This system was validated by crystallizing membrane proteins in lipidic mesophases, including LCP. This system allows for accurate dispensing of LCP material in nanoliter volumes, prevents inadvertent phase transitions that may occur due to dehydration by enclosing LCP in plugs, and is compatible with the traditional method of forming LCP material using a membrane protein sample, as shown by the successful crystallization of bacteriorhodopsin from Halobacterium salinarum. Conditions for the formation of LCP plugs were characterized and presented in a phase diagram. This system was also implemented using two different methods of introducing the membrane protein: 1) the traditional method of generating the LCP material using a membrane protein sample and 2) Post LCP-formation Incorporation (PLI), which involves making LCP material without protein, adding the membrane protein sample externally to the LCP material, and allowing the protein to diffuse into the LCP material or into other lipidic mesophases that may result from phase transitions. Crystals of bacterial photosynthetic reaction centers from Rhodobacter sphaeroides and Blastochloris viridis were obtained using PLI. The plug-based, LCP-assisted microfluidic system, combined with the PLI method for introducing membrane protein into LCP, should be useful for minimizing consumption of samples and broadening the screening of parameter space in membrane protein crystallization.

中文翻译：

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用于分配脂质立方相 (LCP) 材料的基于插头的微流体系统，通过在脂质中间相中结晶膜蛋白进行验证。


本文提出了一种基于塞子的微流体系统，用于分配纳升体积的脂质立方相（LCP）材料塞子，然后将 LCP 塞子与水性塞子合并。该系统通过在脂质中间相（包括 LCP）中结晶膜蛋白进行了验证。该系统可以精确分配纳升体积的 LCP 材料，通过将 LCP 封装在塞子中，防止脱水可能发生的意外相变，并且与使用膜蛋白样品形成 LCP 材料的传统方法兼容，如图所示从盐杆菌中成功结晶细菌视紫红质。对 LCP 塞的形成条件进行了表征并以相图形式呈现。该系统还使用两种不同的引入膜蛋白的方法来实现：1) 使用膜蛋白样品生成 LCP 材料的传统方法和 2) LCP 形成后掺入 (PLI)，其中涉及制备不含蛋白质的 LCP 材料，将膜蛋白样品从外部添加到LCP材料中，并允许蛋白质扩散到LCP材料中或扩散到可能由相变产生的其他脂质中间相中。使用 PLI 获得了球形红杆菌和绿色芽绿藻细菌光合反应中心的晶体。基于插头的LCP辅助微流体系统，与将膜蛋白引入LCP的PLI方法相结合，应有助于最大限度地减少样品消耗并扩大膜蛋白结晶参数空间的筛选。