Label-Free Identification of Single Mononucleotides by Nanoscale Electrophoresis

Nanoscale electrophoresis allows for unique separations of single molecules, such as DNA/RNA nucleobases, and thus has the potential to be used as single molecular sensors for exonuclease sequencing. For this to be envisioned, label-free detection of the nucleotides to determine their electrophoretic mobility (i.e., time-of-flight, TOF) for highly accurate identification must be realized. Here, for the first time a novel nanosensor is shown that allows discriminating four 2-deoxyribonucleoside 5'-monophosphates, dNMPs, molecules in a label-free manner by nanoscale electrophoresis. This is made possible by positioning two sub-10 nm in-plane pores at both ends of a nanochannel column used for nanoscale electrophoresis and measuring the longitudinal transient current during translocation of the molecules. The dual nanopore TOF sensor with 0.5, 1, and 5 µm long nanochannel column lengths discriminates different dNMPs with a mean accuracy of 55, 66, and 94%, respectively. This nanosensor format can broadly be applicable to label-free detection and discrimination of other single molecules, vesicles, and particles by changing the dimensions of the nanochannel column and in-plane nanopores and integrating different pre- and postprocessing units to the nanosensor. This is simple to accomplish because the nanosensor is contained within a fluidic network made in plastic via replication.