A Thermally Responsive Phospholipid Pseudogel: Tunable DNA Sieving with Capillary Electrophoresis

Abstract

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.

Reversible phospholipid nanogels for deoxyribonucleic acid fragment size determinations up to 1500 base pairs and integrated sample stacking

“…Aqueous solutions of the longchain phospholipid dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and the short-chain phospholipid 1,2-dihexanoyl-snglycero-3-phosphocholine (DHPC) are known to form wormlike micelles [19,20]. A dynamic sieving matrix of 10% phospholipid with [DMPC]/[DHPC] = 2.5 was optimized for single-base pair resolution that was adequate for detection of short tandem repeats (STR) in selected loci utilized for human identification using a 100 cm long capillary within a 30 min run time [21]. This is comparable to separations on commercial capillary electrophoresis instruments for forensic STR analyses [22].…”

Aspergillus Collagen-Like Genes ( acl ): Identification, Sequence Polymorphism, and Assessment for PCR-Based Pathogen Detection

“…Linear gels are viscous additives that provide good separation performance and do not need to be polymerized in the capillary. Issues associated with the introduction of highly viscous materials into narrow-bore capillaries have spawned the use of thermally reversible sieving materials (76)(77)(78), including phospholipid nanogels (36). These materials are introduced into the capillary at a temperature that generates low viscosity and then converted into a highly viscous separation additive in the capillary by changing the separation temperature.…”

“…Sieving is then accomplished at 30°C. Separations performed under conditions that do not denature DNA provided nearly single-base resolution of short tandem repeats relevant to human identification (36).…”

“…The accuracy of size standardization is a function of the size of the DNA separated, the available standard size, and the applied voltage. The size range of the measurement affects accuracy, as the DNA migration is linear (i.e., in Ogston sieving) up to ϳ450 bp (36). DNA fragments larger than this undergo reptation-based transport.…”

“…If the precision of the size determination is not critical, capillary nanogel separations can be obtained in shorter separation times (ϳ5 to 10 min) than those shown in Fig. 2C and 4B and yet still yield baseline resolution of the DNA fragments (36).…”