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Open-tubular nanoelectrochromatography (OT-NEC): gel-free separation of single stranded DNAs (ssDNAs) in thermoplastic nanochannels.

ABSTRACT: Electrophoresis or electrochromatography carried out in nanometer columns (width and depth) offers some attractive benefits compared to microscale columns. These advantages include unique separation mechanisms that are scale dependent, fast separation times, and simpler workflow due to the lack of a need for column packing and/or wall coatings to create a stationary phase. We report the use of thermoplastics, in this case PMMA, as the substrate for separating single-stranded DNAs (ssDNAs). Electrophoresis nanochannels were created in PMMA using nanoimprint lithography (NIL), which can produce devices at lower cost and in a higher production mode compared to the fabrication techniques required for glass devices. The nanochannel column in PMMA was successful in separating ssDNAs in free solution that was not possible using microchip electrophoresis in PMMA. The separation could be performed in <1 s with resolution >1.5 when carried out using at an electric field strength of 280 V/cm and an effective column length of 60 μm (100 nm × 100 nm, depth and width). The ssDNAs transport through the PMMA column was driven electrokinetically under the influence of an EOF. The results indicated that the separation was dominated by chromatographic effects using an open tubular nano-electrochromatography (OT-NEC) mode of separation. Interesting to these separations was that no column packing was required nor a wall coating to create the stationary phase; the separation was affected using the native polymer that was UV/O₃ activated and an aqueous buffer mobile phase.

SUBMITTER: Amarasekara CA

PROVIDER: S-EPMC8107830 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:With advances in the design and fabrication of nanofluidic devices during the last decade, there have been a few reports on nucleic acid analysis using nanoscale electrophoresis. The attractive nature of nanofluidics is the unique phenomena associated with this length scale that are not observed using microchip electrophoresis. Many of these effects are surface-related and include electrostatics, surface roughness, van der Waals interactions, hydrogen bonding, and the electric double layer. The majority of reports related to nanoscale electrophoresis have utilized glass-based devices, which are not suitable for broad dissemination into the separation community because of the sophisticated, time consuming, and high-cost fabrication methods required to produce the relevant devices. In this study, we report the use of thermoplastic nanochannels (110 nm x 110 nm, depth x width) for the free solution electrokinetic analysis of ribonucleotide monophosphates (rNMPs). Thermoplastic devices with micro- and nanofluidic networks were fabricated using nanoimprint lithography (NIL) with the structures enclosed via thermal fusion bonding of a cover plate to the fluidic substrate. Unique to this report is that we fabricated devices in cyclic olefin copolymer (COC) that was thermally fusion bonded to a COC cover plate. Results using COC/COC devices were compared to poly(methyl methacrylate), PMMA, devices with a COC cover plate. Our results indicated that at pH = 7.9, the electrophoresis in free solution resulted in an average resolution of the rNMPs >4 (COC/COC device range = 1.94 - 8.88; PMMA/COC device range = 1.4 - 7.8) with some of the rNMPs showing field-dependent electrophoretic mobilities. Baseline separation of the rNMPs was not possible using PMMA- or COC-based microchip electrophoresis. We also found that COC/COC devices could be assembled and UV/O3 activated after device assembly with the dose of the UV/O3 affecting the magnitude of the electroosmotic flow, EOF. In addition, the bond strength between the substrate and cover plate of unmodified COC/COC devices was higher compared to PMMA/COC devices. The large differences in the electrophoretic mobilities of the rNMPs afforded by nanoscale electrophoresis will enable a new single-molecule sequencing platform we envision, which uses molecular-dependent electrophoretic mobilities to identify the constituent rNMPs generated from an intact RNA molecule using a processive exonuclease. With optimized nanoscale electrophoresis, the rNMPs could be identified via mobility matching at an accuracy >99% in both COC/COC and PMMA/COC devices.

Project description:The electrokinetic behavior of molecules in nanochannels (<100?nm in length) have generated interest due to the unique transport properties observed that are not seen in microscale channels. These nanoscale dependent transport properties include transverse electromigration arising from partial electrical double layer overlap, enhanced solute/wall interactions due to the small channel diameter, and field-dependent intermittent motion produced by surface roughness. In this study, the electrokinetic transport properties of deoxynucleotide monophosphates (dNMPs) were investigated, including the effects of electric field strength, surface effects, and composition of the carrier electrolyte (ionic concentration and pH). The dNMPs were labeled with a fluorescent reporter (ATTO 532) to allow tracking of the electrokinetic transport of the dNMPs through a thermoplastic nanochannel fabricated via nanoimprinting (110?nm?×?110?nm, width?×?depth, and 100??m in length). We discovered that the transport properties in plastic nanochannels of the dye-labeled dNMPs produced differences in their apparent mobilities that were not seen using microscale columns. We built histograms for each dNMP from their apparent mobilities under different operating conditions and fit the histograms to Gaussian functions from which the separation resolution could be deduced as a metric to gage the ability to identify the molecule based on their apparent mobility. We found that the resolution ranged from 0.73 to 2.13?at pH?=?8.3. Changing the carrier electrolyte pH?>?10 significantly improved separation resolution (0.80-4.84) and reduced the standard deviation in the Gaussian fit to the apparent mobilities. At low buffer concentrations, decreases in separation resolution and increased standard deviations in Gaussian fits to the apparent mobilities of dNMPs were observed due to the increased thickness of the electric double layer leading to a partial parabolic flow profile. The results secured for the dNMPs in thermoplastic nanochannels revealed a high identification efficiency (>99%) in most cases for the dNMPs due to differences in their apparent mobilities when using nanochannels, which could not be achieved using microscale columns.

Dataset Information

Open-tubular nanoelectrochromatography (OT-NEC): gel-free separation of single stranded DNAs (ssDNAs) in thermoplastic nanochannels.

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