Electrokinetic Separation of Nucleic Acids in Thermoplastic Nanochannels

Abstract

Nucleic acid analysis and separation have recently become an interesting area of research due to their applicability in precision medicine. In precision medicine disease diagnosis, treatment and prevention are tailored to an individual’s genetic makeup. Therefore, there is a huge demand to develop new nucleic acid separation methods that can be utilized in clinical environments. When developing separation methods for clinical applications, it is necessary to consider the growing need for reduced reagent consumption, high throughput analysis, reduced analysis time, sensitive detection and separating nucleic acids in highly complex samples with low analyte concentrations. These factors have limited the use of conventional electrophoretic and chromatographic methods in clinical settings. Therefore, bioanalytical separations have an ever-increasing need for new methods for nucleic acid separations. Development of nucleic acid separation methods for clinical applications are trending toward miniaturization due to low sample and reagent volume consumption, ability to perform high throughput parallel analysis, and unique separation modalities observed at molecular length scales. With the advances in the design and fabrication of nanofluidic devices during the last decade, there have been numerous reports on nucleic acid separations in nanoscale. Nanofluidics offer unique separation modalities that are not observed in microscale due to increased surface interactions such as electrostatic, van de Waals interactions, hydrogen bonding and electric double layer overlap effects. However, the majority of these methods utilize glass-based nanofluidic devices which are not suitable for the use of point of care applications because of sophisticated, time consuming, high cost fabrication methods of these devices. In this study, we report the use of thermoplastic nanochannels (110 nm x 110 nm depth and width, respectively) as an alternative for glass nanochannel devices for the free solution electrokinetic separation of deoxynucleotide monophosphates (dNMPs), ribonucleotide monophosphates (rNMPs) and short single stranded DNA. Thermoplastic nanofluidic devices with mixed scale micro and nanofluidic networks were fabricated using a simple, high resolution nanoimprinting lithography method and the structures were enclosed via thermal fusion bonding to a cover plate. We were able to separate ss-DNA in these thermoplastic nanochannels without any additives to the buffer and we found that the separation mechanism is electrochromatographic. In the separation of rNMPs and dNMPs we found that the separation is majorly electrophoretic but, with some impact on the separation by surface interactions of these analytes with nanochannel walls.