(9c) Isolation and Concentration of Biomarkers Using Self-Assembled Nanomaterials

As methods to investigate and model biological systems grow in complexity, there is an increasing need to isolate and concentrate biomarkers from biological fluids, environmental samples, and tissue sections. Biomarkers, including DNA, RNA, and proteins can only be recognized by specific affinity interactions between the biomarker and some ligand. Typical affinity separations utilize ligands immobilized onto a solid support such as a microbead or resin. In many cases, these surfaces are prone to fouling by the vast excess of non-target proteins, lipids, and other adventitious material that can adsorb and deactivate affinity ligands. Taking a cue from biological recognition processes that occur at cell membranes, we show that the soft surfaces presented by micelles and liposomes are less prone to biofouling since nonpolar moieties are sequestered into the nonpolar interior of these self-assembled structures.

The use of self-assembled nanomaterials for separations has other advantages as well. Electrosteric effects dictate that DNA hybridization events occur more slowly and with lower yield when one of the strands is anchored to a solid surface. Properly engineered systems have the affinity ligand bind in solution, prior to self-assembly of the bound complex with its carrier micelle. Such systems also are compatible with overhanging stretches of unbound DNA that would sterically clash with a solid surface. This opens the possibility of isolating DNA directly from sheared biological samples, without the use of PCR or restriction digests that would give a known terminal sequence.

Work in our lab that implements this “tag-and-assemble” approach to purify DNA targets using open-channel electrophoresis. Affinity tags are composed of peptide nucleic acid (PNA), a synthetic DNA mimic, linked to alkane chains of varying length. Using the method, single- and double-stranded DNA oligomers up to 1000 bases in length can be purified, even in the presence of large amounts of serum protein. Partitioning of the PNA-DNA complex to micelles is strongly dependent on the alkane chain length, making possible the simultaneous purification of multiple targets by linking probe sequences to different chain lengths. We will also discuss our efforts to purify proteins using DNA aptamer-surfactants co-assembled with solvent-swollen microemulsions and large unilamellar vesicles.