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

Abstract

Phospholipid additives are a cost-effective medium to separate deoxyribonucleic acid (DNA) fragments and possess a thermally-responsive viscosity. This provides a mechanism to easily create and replace a highly viscous nanogel in a narrow bore capillary with only a 10°C change in temperature. Preparations composed of dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) self-assemble, forming structures such as nanodisks and wormlike micelles. Factors that influence the morphology of a particular DMPC-DHPC preparation include the concentration of lipid in solution, the temperature, and the ratio of DMPC and DHPC. It has previously been established that an aqueous solution containing 10% phospholipid with a ratio of [DMPC]/[DHPC]=2.5 separates DNA fragments with nearly single base resolution for DNA fragments up to 500 base pairs in length, but beyond this size the resolution decreases dramatically. A new DMPC-DHPC medium is developed to effectively separate and size DNA fragments up to 1500 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 1500 base pairs. This increase in the upper size limit is accomplished using commercially available phospholipids at an even lower material cost than is achieved with the 10% preparation. The separation additive is used to evaluate size markers ranging between 200 and 1500 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 self-assembled 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.

Capillary electrophoresis with stationary nanogel zones of galactosidase and Erythrina cristagalli lectin for the determination of β(1–3)-linked galactose in glycans

“…Following enzyme treatment the product was then separated in the remaining portion of the capillary. In prior reports the capillary was filled with nanogel to improve the separation;[21, 24, 25, 31, 32] however, when higher resolution was not required the stationary zone of enzyme functioned equally well when it was introduced into a capillary filled with aqueous background electrolyte instead of nanogel. [24]…”

Orthogonal Tip-to-Tip Nanocapillary Alignment Allows for Easy Detection of Fluorescent Emitters in Femtomolar Concentrations

“…45 Furthermore, we expect that attomolar molecule detection might be realized in the case that one uses preconcentration methods like sweeping, 46 electric field amplification, 39,47 or sample stacking by nanogel. 48 Thus, our method is not only beneficial to the ultrasensitive analysis of essential molecules but also potentially helpful for the determination of trace circulating biomarkers in various body fluids.…”

Recent progress of sample stacking in capillary electrophoresis (2014–2016)

“…Durney et al. proposed a novel stacking principle for the improvement of DNA separation, based on phospholipid nanogels with highly temperature dependent viscosity. The separation gel (forming the BGE) was formed by 100 mM MOPS at pH 7.0 containing 2.5 % w/v of dimyristoyl‐sn‐glycero‐3‐phosphocholine and 1,2‐dihexanoyl‐sn‐glycero‐3‐phosphocholine (5:2).…”

“…The process of discontinuous phospholipid nanogels in which the standard DNA mixture is stacked in the 10% phospholipid and separated in the 2.5% phospholipid (for explanation, see text). Reprinted from with permission from Elsevier.…”