Detection and identification of single ribonucleotide monophosphates using a dual in-plane nanopore sensor made in a thermoplastic via replication.

We report the generation of ∼8 nm dual in-plane pores fabricated in a thermoplastic via nanoimprint lithography (NIL). These pores were connected in series with nanochannels, one of which served as a flight tube to allow the identification of single molecules based on their molecular-dependent apparent mobilities (i.e., dual in-plane nanopore sensor). Two different thermoplastics were investigated including poly(methyl methacrylate), PMMA, and cyclic olefin polymer, COP, as the substrate for the sensor both of which were sealed using a low glass transition cover plate (cyclic olefin co-polymer, COC) that could be thermally fusion bonded to the PMMA or COP substrate at a temperature minimizing nanostructure deformation. Unique to these dual in-plane nanopore sensors was two pores flanking each side of the nanometer flight tube (50 × 50 nm, width × depth) that was 10 µm in length. The utility of this dual in-plane nanopore sensor was evaluated to not only detect, but also identify single ribonucleotide monophosphates (rNMPs) by using the travel time (time-of-flight, ToF), the resistive pulse event amplitude, and the dwell time. In spite of the relatively large size of these in-plane pores (∼8 nm effective diameter), we could detect via resistive pulse sensing (RPS) single rNMP molecules at a mass load of 3.9 fg, which was ascribed to the unique structural features of the nanofluidic network and the use of a thermoplastic with low relative dielectric constants, which resulted in a low RMS noise level in the open pore current. Our data indicated that the identification accuracy of individual rNMPs was high, which was ascribed to an improved chromatographic contribution to the nano-electrophoresis apparent mobility. With the ToF data only, the identification accuracy was 98.3%. However, when incorporating the resistive pulse sensing event amplitude and dwell time in conjunction with the ToF and analyzed via principal component analysis (PCA), the identification accuracy reached 100%. These findings pave the way for the realization of a novel chip-based single-molecule RNA sequencing technology.

Nanofluidic devices for the separation of biomolecules.

Over the last 30-years, microchip electrophoresis and its applications have expanded due to the benefits it offers. Nanochip electrophoresis, on the other hand, is viewed as an evolving area of electrophoresis because it offers some unique advantages not associated with microchip electrophoresis. These advantages arise from unique phenomena that occur in the nanometer domain not readily apparent in the microscale domain due to scale-dependent effects. Scale-dependent effects associated with nanochip electrophoresis includes high surface area-to-volume ratio, electrical double layer overlap generating parabolic flow even for electrokinetic pumping, concentration polarization, transverse electromigration, surface charge dominating flow, and surface roughness. Nanochip electrophoresis devices consist of channels with dimensions ranging from 1 to 1000 nm including classical (1-100 nm) and extended (100 nm - 1000 nm) nanoscale devices. In this review, we highlight scale-dependent phenomena associated with nanochip electrophoresis and the utilization of those phenomena to provide unique biomolecular separations that are not possible with microchip electrophoresis. We will also review the range of materials used for nanoscale separations and the implication of material choice for the top-down fabrication and operation of these devices. We will also provide application examples of nanochip electrophoresis for biomolecule separations with an emphasis on nano-electrophoresis (nEP) and nano-electrochromatography (nEC).