Novel force spectroscopy with nanopores

Nanopores are not only basic building blocks in every living organism but emerging tools for the characterisation of single molecules in aqueous solution. The main probe for detection of a molecule in a nanopore is the ionic current driven by an external voltage. A brief change in conductance indicates the passage of a single molecule through the membrane containing the pore. Until now, nanopores were successfully used to detect the presence DNA, RNA, proteins and even single ions. There are two main sources for nanopores. The top-down approach employs nanotechnology and highly focussed electron beams to fabricate single pores in insulating membranes. The bottom-up possibility is to extract biological channels from bacteria and use them as sensors embedded in a native lipid membrane. The aim of this proposal is to use solid-state interfaced with biological nanopores to develop a novel force spectroscopy on the single molecule level. Nanopores offer a unique possibility for analysis of molecules in a minute volume and studies of single molecules in strong confinement. Electric fields allow to pull charged macromolecules into and through the pore, while the ion current contains information about the molecule charge, diameter, length and its interaction with the channel surface. Here we use optical tweezers with ion current detection to apply forces and accurately position a molecule in a pore. We will genetically modify the biological nanopores to study the influence of interactions on the translocation time and forces. This will lead to a novel, nanopore-based force spectroscopy enabling pico-Newton force detection while controlling the distance with nanometer resolution along a molecule in a biological nanopore. Our tool will be used to investigate the underlying physics of interaction, translocation or unfolding of macromolecules in pores. The current consortium will claim a worldwide lead position with this technique. Our proposed technology could lead to groundbreaking experiments in the areas of biological physics, biochemistry and biology.