Pure and Applied Biochemistry, Center for Chemistry and Chemical Engineering, Lund University, Lund, Sweden.
Electrophoresis. 2010 Jan;31(3):459-64. doi: 10.1002/elps.200900464.
Totally porous lipid-based liquid crystalline nanoparticles were used as pseudostationary phase for capillary electroseparation with LIF detection of proteins at physiological conditions using unmodified cyclic olefin copolymer capillaries (Topas, 6.7 cm effective length). In the absence of nanoparticles, i.e. in CE mode, the protein samples adsorbed completely to the capillary walls and could not be recovered. In contrast, nanoparticle-based capillary electroseparation resolved green fluorescent protein from several of its impurities within 1 min. Furthermore, a mixture of native green fluorescent protein and two of its single-amino-acid-substituted variants was separated within 2.5 min with efficiencies of 400 000 plates/m. The nanoparticles prevent adsorption by introducing a large interacting surface and by obstructing the attachment of the protein to the capillary wall. A one-step procedure based on self-assembly of lipids was used to prepare the nanoparticles, which benefit from their biocompatibility and suspension stability at high concentrations. An aqueous tricine buffer at pH 7.5 containing lipid-based nanoparticles (2% w/w) was used as electrolyte, enabling separation at protein friendly conditions. The developed capillary-based method facilitates future electrochromatography of proteins on polymer-based microchips under physiological conditions and enables the initial optimization of separation conditions in parallel to the chip development.
全多孔脂质基液晶纳米颗粒被用作伪固定相，用于在生理条件下使用未改性环状烯烃共聚物毛细管（Topas，有效长度 6.7 厘米）进行带有 LIF 检测的蛋白质胶束电动色谱分离。在没有纳米颗粒的情况下，即在 CE 模式下，蛋白质样品完全吸附在毛细管壁上，无法回收。相比之下，基于纳米颗粒的毛细管电动分离在 1 分钟内从几种杂质中分辨出绿色荧光蛋白。此外，在 2.5 分钟内，天然绿色荧光蛋白及其两种单氨基酸取代变体的混合物以 400000 板/m 的效率分离。纳米颗粒通过引入大的相互作用表面并阻止蛋白质与毛细管壁的附着来防止吸附。基于脂质自组装的一步法用于制备纳米颗粒，其得益于其生物相容性和在高浓度下的悬浮稳定性。在 pH 7.5 的三羟甲基氨基甲烷缓冲液中含有基于脂质的纳米颗粒（2%w/w）用作电解质，可在蛋白质友好的条件下进行分离。开发的基于毛细管的方法促进了未来在生理条件下在聚合物基微芯片上的蛋白质电色谱分离，并能够在芯片开发的同时并行优化分离条件。
