Clogging Probability of Nicked DNA Molecules on Solid-State Nanopores

Abstract

Solid state nanopores can detect single DNA molecules with sub-molecule resolution by measuring ion current variations when a molecule passes through a nanopore. However, DNA molecules frequently clog at a pore even they enter individually, particularly DNA molecules are relatively long such as lambda DNA (48.5kbp), T4 DNA (166kbp). We are interested in strong interactions of DNA with pores against the electrophoretic force that causes clogging. We assumed the cause of clogging is electroosmosis not only by the surface of pore wall but also DNA itself since negatively charged DNA attracts positive ions, which generates counterflow for DNA translocation. To enhance the flow by electroosmosis, we used nicked DNA molecules since more flexible nicked DNA likely form more folding configuration inside a pore, which induces higher flow rate of electroosmosis. Focused ion beams were used on Si3N4 (200 nm) thin films to create nanopores with a diameter of 100 and 200 nm. lambda DNA (48.5kbp), T4 DNA (166kbp) were chosen for DNA and three types of restriction enzymes, Nb.BsmI, Nb.BssSI and Nt.BstNBI, were used for nick density variation. DNA was directly observed with a fluorescent molecule (YOYO-1) on an optical microscope. We found that clogging probabilities increases with nicks especially for lambda DNA. In addition, more nicks increased the clogging probability for both lambda DNA and T4 DNA. Interestingly, we observed that nicked DNA molecules released from a pore to the cis side after it captured as clogging. Such DNA returned to a pore electrophoretically for its translocation. To evaluate the forces by electroosmosis and electrophoresis quantitatively, a finite element analysis was performed. Finally, we discuss an application of a nanopore devise for filtering nicked DNA molecules.