Diamond Cubic Phase of Monoolein and Water as an Amphiphilic Matrix for Electrophoresis of Oligonucleotides Nils Carlsson, † Ann-Sofie Winge, † Sven Engstro 1 m, ‡ and Bjo 1 rn Åkerman* ,† Department of Chemistry and Bioscience and Department of Materials and Surface Chemistry, Chalmers UniVersity of Technology, S412 96 Go ¨teborg, Sweden ReceiVed: April 3, 2005; In Final Form: July 21, 2005 We used a cubic liquid crystal formed by the nonionic monoglyceride monoolein and water as a porous matrix for the electrophoresis of oligonucleotides. The diamond cubic phase is thermodynamically stable when in contact with a water-rich phase, which we exploit to run the electrophoresis in the useful submarine mode. Oligonucleotides are separated according to size and secondary structure by migration through the space-filling aqueous nanometer pores of the regular liquid crystal, but the comparatively slow migration means the cubic phase will not be a replacement for the conventional DNA gels. However, our demonstration that the cubic phase can be used in submarine electrophoresis opens up the possibility for a new matrix for electrophoresis of amphiphilic molecules. From this perspective, the results on the oligonucleotides show that water-soluble particles of nanometer size, typical for the hydrophilic parts of membrane-bound proteins, may be a useful separation motif. A charged contamination in the commercial sample of monoolein, most likely oleic acid that arises from its hydrolysis, restricts useful buffer conditions to a pH below 5.6. Introduction The basic mechanism of electrophoretic DNA migration in hydrogels such as agarose and polyacrylamide is well- understood. 1,2 However, quantitative modeling of the migration is limited to statistical descriptions of the gel structure, because the underlying gelation mechanisms are stochastic in nature, such as phase separation in agarose and free-radical polymer- ization for polyacrylamide. One approach to obtain more well- defined matrixes for electrophoresis is to use lithography to create obstacle courses of pillars 3 or plateaus 4 sandwiched between two planar surfaces. These structures have been important for improving our understanding of the confined migration of DNA but, to date, have not found major separation applications as a result of their low sample capacity. Another approach is to use amphiphilic lipids or polymers, which together with water self-assemble into lamellar, hexagonal, or cubic phases which often have gel-like properties, albeit being pastier than the solid nature of the conventional hydrogels. These well-ordered liquid crystals contain nanometer aqueous channels confined between micelles or membranes formed by the amphiphile, although they tend to be polycrystalline on mac- roscopic length scales. One interesting example is the cubic phase that results from the packing of discrete micelles formed in water by the Pluronic F127 block copolymer of poly(ethylene oxide) and poly- (propylene oxide), where the aqueous channels between the micelles are of nanometer dimensions. This structure can be used as a molecular sieve for DNA separation, 8,9 although larger plasmid-sized DNAs do not enter the pores of the cubic phase but rather migrate along grain boundaries in the polycrystalline structure. 5,6 The F127 system has a reversed thermal dependence compared to that of agarose in that it forms a flowing solution at low temperatures (around 10 °C) but turns to the gel-like cubic phase at room temperature. 7-9 This transformation is reversible, which allows for easy filling and emptying in capillary electrophoresis 10,11 and for in situ polymerase chain reaction in slab-gel electrophoresis. 6 A drawback with the phase behavior of the Pluronic system is that its cubic phase cannot be in equilibrium with water. This means that the Pluronic gels tend to dissolve in the parts that are in contact with the buffer chambers that contain the electrodes. 5 In particular, it is not possible to employ the useful submarine mode of electrophoresis where the gel is covered by buffer to maintain its ionic composition. Here, we try to circumvent this inherent limitation of the Pluronic system by choosing a cubic structure formed by the monoglyceride monoolein (Chart 1) and water. 12 The diamond-type monoolein cubic phase can be in equilibrium with a water-rich phase, 13,14 which has the potential to act as a buffer reservoir. The first aim of this study was to investigate if the diamond cubic phase can be used in submarine electrophoresis. In addition to their well-ordered structure, liquid crystals have an even more interesting advantage over conventional hydrogels in that their amphiphilic nature may allow the analysis of amphiphilic analytes. In this perspective, the monoolein cubic phase differs from the F127 packed micelle type in an important aspect. Monoolein and water form a bicontinuous structure where the lipid bilayer membrane, which lines the space-filling system of aqueous pores, is continuous too, whereas in the F127 micellar cubic phase, the hydrophobic part is discrete. The monoolein cubic phase, thus, allows the transport of membrane- * Corresponding author. † Department of Chemistry and Bioscience. ‡ Department of Materials and Surface Chemistry. CHART 1 18628 J. Phys. Chem. B 2005, 109, 18628-18636 10.1021/jp0516893 CCC: $30.25 © 2005 American Chemical Society Published on Web 09/08/2005