Microfluidic device for coupling isotachophoretic sample focusing with nanopore single-molecule sensing

Solid-state nanopores (NPs) are label-free single-molecule sensors, capable of performing highly sensitive assays from a small number of biomolecule translocation events. However, single-molecule sensing is challenging at extremely low analyte concentrations due to the limited flux of analytes to the sensing volume. This leads to a low event rate and increases the overall assay time. In this work, we present a method to enhance the event rate at low analyte concentrations by using isotachophoresis (ITP) to focus and deliver analytes to a nanopore sensor. Central to this method is a device capable of performing ITP focusing directly on a solid-state NP chip, while preventing the focusing electric field from damaging the nanopore membrane. We discuss considerations and trade-offs related to the design of the focusing channel, the ITP electrolyte system and electrical decoupling between the focusing and sensing modes. Finally, we demonstrate an integrated device wherein the concentration enhancement due to ITP focusing leads to an increase in event rate of >300-fold in the ITP-NP device as compared to the NP-only case.