Reversible Phospholipid Nanogels: An Alternative Matrix for Tunable High Resolution DNA Sieving with Enhanced Separation Efficiency and Accurate Extended Range Sizing by Capillary Gel 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., pseudo-gels) 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. While a practical advantage of utilizing phospholipid pseudo-gels for sieving is the ease with which they are introduced into the separation capillary, the separation performance of the material for DNA analyses is exceptional. Capillary electrophoresis separations of DNA are achieved with separation efficiencies ranging from 400,000 to 7,000,000 theoretical plates in a 25 micrometer inner diameter fused silica capillary. Assessment of the phospholipid nanogel 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. Single base resolution of short tandem repeats relevant to human identification is accomplished with 30 minute 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 pseudo-gel preparation described here provides a new innovation to sieving based capillary separations. Specific DMPC-DHPC medium is developed to effectively separate and size DNA fragments up to 1,500 base pairs by decreasing the total lipid concentration to 2.5%. A 2.5% phospholipid nanogel generates a resolution of 1% of the DNA fragment size up to 1,500 base pairs. This separation additive is used to evaluate size markers ranging between 200 and 1,500 base pairs in order to distinguish invasive strains of Streptococcus pyogenes and Aspergillus species by harnessing differences in gene sequences of collagen-like proteins in these organisms. For the first time, a reversible stacking gel is integrated in a capillary sieving separation by utilizing the thermally-responsive viscosity of these selfassembled phospholipid preparations. A discontinuous matrix is created that is composed of a cartridge of highly viscous phospholipid assimilated into a separation matrix of low viscosity. DNA sample stacking is facilitated with longer injection times without sacrificing separation efficiency