Translocation Kinetics of DNA through Nanopores Interfaced with Agarose Gel

Abstract

Nanopores have recently emerged as an exciting tool for the study of single biological molecules. Essential to many nanopore-based applications, including DNA sequencing, is the ability to control the motion of charged biomolecules through the pore. While previous studies have primarily focused on increasing the average translocation times by changing the salt concentration, counter ion type, etc., velocity fluctuations during passage of identical DNA molecules remains a limitation for many applications.Here, the aim is to reduce the spread in the distribution of translocation times by controlling the initial conformation of DNA molecules captured by a solid-state nanopore. The nanopores are fabricated on a silicon nitride membrane with agarose gel on the trans side, essentially covering the membrane in a matrix of agarose fiber bundles. The DNA injected into the solution on the cis side passes through the nanopore and becomes trapped within the pores of the agarose. Once in the gel, the geometry of the DNA is limited and each molecule is forced to adopt a similar conformation. We compare the translocation kinetics under both voltage polarities, either driving DNA from the solution to the gel or vice versa. Furthermore, the distribution of translocation times is studied as a function of the agarose pore size, which is tuneable (300nm - 1μm), and dsDNA fragment lengths, allowing us to probe various conformational states. This investigation of the translocation kinetics of DNA through solid-state nanopores when captured from a gel matrix will lead to a better understanding of electrophoretic motion of dsDNA molecules through nanoconfined geometries and may provide a path to not only control the distribution of translocation times, but also reduce the passage speed.