Investigation in to the Use of Thermoplastic Nanochannels for Time of Flight (TOF) Detection of Nucleotide Monophosphates: Towards Single Molecule DNA Sequencing

Because of the unique properties that arise when the column size is comparable to either the length scale of electrostatic interactions or the size of the molecules being transported through them, nanochannel-based devices have garnered attention for many applications, especially nanoelectrophoresis. One essential application looking to exploit unique phenomena that occur at the nanoscale is Single Molecule Sequencing (SMS). SMS offers advantages over conventional ensemble-based sequencing platforms. Our proposed SMS device looks to identify nucleotides based on their molecular-dependent flight times as they migrate through a nanochannel, termed Time-of-Flight (ToF) detection. This research looked to understand the use of thermoplastic substrates for the fabrication of nanochannels to utilize in nanoelectrophoresis experiments for ToF detection. Differences in the migration properties of dNMPs under varying pH, buffer additives and buffer concentration to enhance the resolution of the separation and ultimately result in a high base calling accuracy were explored as well. Super Resolution Fluorescence data indicated non-uniform distributions of -COOH functional groups for both COC and PMMA thermoplastics with the degree of heterogeneity being dose dependent. In addition, COC demonstrated relative higher surface density of functional groups compared to PMMA for both UV/O3 and O2 plasma treatment. The spatial distribution of -COOH groups secured from super-resolution imaging were used to simulate non-uniform patterns of electroosmotic flow in thermoplastic nanochannels. Simulations were compared to single-particle tracking of fluorescent nanoparticles within thermoplastic nanoslits to demonstrate the effects of surface functional group heterogeneity on the electrokinetic transport process. Furthermore, results showed that increased norbornene content within COC led to the generation of more oxygen containing functionalities such as alcohols, ketones, aldehydes and carboxyl groups when activated with either UV/O3 or O2 plasma. Specifically, COC 6017 (~60% norbornene content) showed a significantly higher –COOH functional group density when compared to COC 6013 (~50% norbornene content) and COC 8007 (~35% norbornene content) following UV/O3 or O2 plasma activation. Furthermore, COC 6017 showed a smaller average RMS roughness (0.65 nm) when compared to COC 8007 (0.95 nm) following activation making this substrate especially suited for nanofluidic applications, which require smooth surfaces to minimize effects arising from dielectrophoretic trapping or non-specific adsorption. Although all COC substrates showed >90% transparency at wavelengths >475 nm, COC 6017 showed significantly less transparency at wavelengths below 475 nm following activation, making optical detection in this region difficult. Our data showed distinct physiochemical differences in activated COC that was dependent upon the ethylene/norbornene content of the thermoplastic and thus, careful selection of the particular COC grade must be considered for micro- and nanofluidics. Finally, we determined that nanoscale columns introduce unique surface interactions differences of the dNMPs allowing for resolutions ranging from 0.42-0.94 and changes in the pH that can further enhance resolutions up to 2.7. Furthermore, it was determined that low buffer concentrations resulting in EDL overlap decrease the resolution. In addition, nanoscale electrophoresis was performed on the sub-second time scale, resulting in highly efficient separations. Ultimately, our research shows great promise for the use of nanoelectrophoresis within thermoplastic columns for the separation of dNMPs among many other molecules, not achievable on the microscale.Doctor of Philosoph