Tailoring Thermoplastic In-Plane Nanopore Size by Thermal Fusion Bonding for the Analysis of Single Molecules

We report a simple method for tailoring the size of in-plane nanopores fabricated in thermoplastics for single-molecule sensing. The in-plane pores were fabricated via nanoimprint lithography (NIL) from resin stamps, which were generated from Si masters. We could reduce the size of the in-plane nanopores from 30 to ∼10 nm during the thermal fusion bonding (TFB) step, which places a cover plate over the imprinted polymer substrate under a controlled pressure and temperature to form the relevant nanofluidic devices. Increased pressures during TFB caused the cross-sectional area of the in-plane pore to be reduced. The in-plane nanopores prepared with different TFB pressures were utilized to detect single-λ-DNA molecules via resistive pulse sensing, which showed a higher current amplitude in devices bonded at higher pressures. Using this method, we also show the ability to tune the pore size to detect single-stranded (ss) RNA molecules and single ribonucleotide adenosine monophosphate (rAMP). However, due to the small size of the pores required for detection of the ssRNA and rAMPs, the surface charge arising from carboxylate groups generated during O₂ plasma oxidation of the surfaces of the nanopores to make them wettable had to be reduced to allow translocation of coions. This was accomplished using EDC/NHS coupling chemistry and ethanolamine. This simple modification chemistry increased the event frequency from ∼1 s^-1 to >136 s^-1 for an ssRNA concentration of 100 nM.