Enhancing Resolution In Microchip DNA Electrophoresis by Tailoring Hydrogel Nanostructure to Exploit Entropic Trapping

Electrophoretic DNA transport through nanoporous hydrogels is characterized by an entropic trapping (ET) mechanism when the DNA size is close to the gel pore size. This mode of transport can yield highly desirable transport properties (e.g., enhanced size dependences of mobility and diffusion for improved separations). But hydrogels have largely been ignored in efforts to understand and exploit ET effects, where the focus has instead centered on simpler idealized ?nanofilter? architectures based on arrays of alternating deep wells and narrow slits (i.e., describable by only two length scales).

We have recently developed a new model that captures the inherently heterogeneous pore morphology comprising realistic hydrogel matrices, enabling key features of macromolecular transport in the ET regime to be successfully predicted. When combined with material characterization methods we have developed that enable both the mean pore size and pore size distribution of the gel to be quantified, we are able to establish a direct link between sieving matrix morphology and DNA migration in photopolymerized crosslinked polyacrylamide gels cast under different UV intensities and concentrations. We can then directly apply these insights using a microfluidic platform that allows continuous whole channel scanning of DNA separation progress so that the size dependence of these parameters can be quickly and accurately measured. A photocurable hydrogel matrix allows the pore architecture to be tailored by adjusting the UV light intensity. In this way, we are able to select polymerization conditions that produce pore morphologies favorable for ET, and harness these effects to achieve improved separation performance.

We have applied this approach to examine electrophoresis of double-stranded DNA in the 100 to 1000 bp size range, and have identified gel polymerization conditions that induce a transition in the physical mechanism of DNA migration from reptation to entropic trapping. Furthermore, we have identified a range of conditions where a favorable interplay exists between these migration mechanisms that results in improved separation performance (e.g., resolving power that increases with DNA size, the opposite of what is conventionally observed). These new insights have also led to an unexpected prediction that oscillating the applied electric field at a specific frequency, in a specific gel morphology where ET effects dominate, can establish a resonance phenomenon that greatly improves separation resolution by reducing diffusion. Here the electric field is generated in an ?on-off? manner to synchronize discrete hops between neighboring pores in a very specific gel morphology, in contrast to conventional pulsed field methods. Our microchip electrophoresis experiments support this prediction, with a doubling in separation resolution observed at DNA fragment sizes below 500 bp.