Electrophoretic processes have long been instrumental in the identification of biomolecules; to obtain length and sequence data for DNA and RNA, and to identify proteins by their molecular weight and isoelectric point. Generally, electrophoresis is performed in agarose or poly(acrylamide) sieving matrix to discriminate biomolecules by their molecular weight and charge. These methods are also compatible with a host of labeling methods to more specifically identify biomolecules, and have been miniaturized to capillary and microchip electrophoresis formats for automated, high-throughput analysis. Despite their widespread application, the use of polymeric sieving matrices requires fairly extensive pre-purification of samples to avoid clogging and deposition of adventitious material. Multiple, 2-D separations are generally required to obtain both molecular weight and charge data, and in most cases probes must be incorporated by post-separation blotting methods, or their direct linkage to the matrix.

We present a one-step approach for the separation and analysis of DNA, RNA, and proteins using surfactant-like affinity tags. The tags are oligomers of DNA, RNA, or nucleic acid analogues conjugated to n-alkanes of varying length (C12-C18). Minimally processed samples are treated with the tags to allow for specific binding to targets, and are then injected into the uncoated capillary of a commercial capillary electrophoresis system pre-loaded with a buffer containing micelles of the non-ionic surfactant Triton X-100. On the application of an electric field, electro-osmotic flow flushes the tagged sample with micelles, which bind the tagged molecules conferring a shift in their mobility that separates them from untagged components. Importantly, the tagged molecules do not irreversibly bind to the micelles; instead, they partition to the micelles with a partition coefficient that reveals the size and charge of the tagged species. As such, a single electropherogram can provide separation as well as characterization of an analyte of interest. Additionally, since separations are performed in the presence of concentrated surfactant solutions, adventitious material that would otherwise foul uncoated capillaries and sieving matrices is taken up by the micelles with minimal impact on the separation fidelity.

This talk will describe three applications of this surfactant-based separation strategy. One is the identification of DNA oligomers using amphiphilic tags composed of the nucleic acid analog peptide nucleic acid (PNA). We demonstrate that oligomers up to 1000 bases in length can be separated from untagged DNA using a single C18 amphiphilic tag. Both the partitioning of the tagged DNA to Triton X-100 and the mobility of the micelle/DNA complex are strong functions of the molecular weight and therefore can be use to ascertain the molecular weight of an unknown sample. Partitioning is extremely sensitive to the length of alkane used (C12-C18), and this can be leveraged for separation of multiple targets in a single run. The use of multiple tags on a single analyte can also be used to extend the range of molecular weight that can be discriminated. Since these separations can be performed in an uncoated capillary, the electro-osmotic flow can be tuned to discriminate oligomers with very small differences in molecular weight. We will also describe DNA sequencing methods that do not require sieving matrices, using the micelles as “drag-tags.” Micelles have advantages over other types of drag-tags, such as proteins or polymers. One is that in the presence of high electric fields, the tagged DNA can release from its micelle without losing its tag, leaving it free to interact with other micelles in the system. Finally, we will also discuss our preliminary efforts to simultaneously separate and characterize peptides and proteins using aptamer-surfactants as affinity tags in micellar electrokinetic separations.

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