A Thermally
Responsive Phospholipid Pseudogel: Tunable
DNA Sieving with Capillary Electrophoresis
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Abstract
In an aqueous solution the phospholipids
dimyristoyl-<i>sn</i>-glycero-3-phosphocholine (DMPC) and
1,2-dihexanoyl-<i>sn</i>-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