A thermally responsive phospholipid pseudogel: tunable DNA sieving with capillary electrophoresis

In an aqueous solution the phospholipids dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) self-assemble to form thermo-responsive non-Newtonian fluids (i.e., pseudogels) in which small temperature changes of 5-6 °C decrease viscosity dramatically. This characteristic is useful for sieving-based electrophoretic separations (e.g., of DNA), as the high viscosity of linear sieving additives, such as linear polyacrylamide or polyethylene oxide, hinders the introduction and replacement of the sieving agent in microscale channels. Advantages of utilizing phospholipid pseudogels for sieving are the ease with which they are introduced into the separation channel and the potential to implement gradient separations. Capillary electrophoresis separations of DNA are achieved with separation efficiencies ranging from 400,000 to 7,000,000 theoretical plates in a 25 μm i.d. fused silica capillary. Assessment of the phospholipid pseudogel with a Ferguson plot yields an apparent pore size of ~31 nm. Under isothermal conditions, Ogston sieving is achieved for DNA fragments smaller than 500 base pairs, whereas reptation-based transport occurs for DNA fragments larger than 500 base pairs. Nearly single base resolution of short tandem repeats relevant to human identification is accomplished with 30 min separations using traditional capillary electrophoresis instrumentation. Applications that do not require single base resolution are completed with faster separation times. This is demonstrated for a multiplex assay of biallelic single nucleotide polymorphisms relevant to warfarin sensitivity. The thermo-responsive pseudogel preparation described here provides a new innovation to sieving-based capillary separations.