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.

Keywords: DNA; Electrochromatography; Nanofluidics; Thermoplastics.

Figure 1.

(A) An image of the T-chip used for the microscale electrophoresis. The chip was made via hot embossing into PMMA. (B) Schematic diagram of the in-house built microchip electrophoresis laser-induced fluorescence detector that utilized a 20 mW, 532 nm excitation laser with edge filter. The detector contained a 560 nm long pass filter, 532 nm dichroic filter and SPCM-AQR single photon counting module within the optical train. A 100× high numerical aperture (NA = 1.3) microscope objective was used to focus the laser beam onto the microchannel and collect the fluorescence.

Figure 2.

SEM images of the Si master, resin stamp, and imprinted device. (A) SEM of the Si master at 500X magnification. (B) Enlarged area of the entrance funnel formed in the Si master. (C) UV-imprinted nanochannel device to form the resin stamp with PUA. (D) Enlarged nanochannel area in the resin stamp. (E) Nanochannels thermally imprinted into a PMMA substrate. (F) Enlarged area of nanochannel imprinted into PMMA.

Figure 3.

(A) Schematic diagram of experimental set up used for the microscale electrophoresis, where a T-shaped microchip was used. A sample plug was electrokinetically introduced into the separation channel by applying a potential across the S and SW reservoirs. A photon avalanche detector was used to capture the fluorescence signal at the detection point. (B) Electropherogram obtained for the free solution microchip (PMMA) electrophoresis of ssDNAs with microchannel dimensions of 50 μm × 100 μm (depth and width, respectively) with a length of 5.0 cm. The applied voltage for the electrophoresis was 5 kV (1000 V/cm) with a 45 mM TBE buffer (pH 8.3) used as the background electrolyte. The ssDNAs (5 μM) were injected electrokinetically into the separation channel.

Figure 4.

Characterization of surface roughness by AFM for IM-PMMA and NIM-PMMA surfaces before and after O₂ plasma activation. Shown are AFM images of: (A) IM-PMMA; (B) NIM-PMMA; (C) O₂ plasma treated IM-PMMA; and (D) O₂ plasma treated NIM PMMA. Plasma activation was done at 50 mW for 1 min. These images were taken by scanning an area of 3 μm × 3 μm. (E) Comparison of the measured root mean square (RMS) roughness of both PMMA types before and after O₂ plasma activation. (F) The measured EOF for NIM-PMMA and IM-PMMA nanochannel devices as well as the zeta potential following O2 plasma activation (EOF measured at pH = 8.3).

Figure 5.

(A) Effective mobility of oligonucleotides versus the field strength in NIM-PMMA (substrate) – COC 8007 (cover plate) nanochannels (100 nm depth × 100 nm width and 60 μm effective length). (B) Effective mobility of ssDNAs versus electric field strength in IM-PMMA (substrate)-COC 8007 (cover plate) nanochannels (100 nm depth × 100 nm width and 60 nm effective length). Note: the lines in (A) and (B) are shown following trends and are not intended to indicate a functional fit to the data point. (C) Semi log plot of effective mobility versus length of ssDNAs in NIM-PMMA and IM-PMMA at 280 V/cm.

Figure 6.

(A) Histogram of effective migration time of Oligo 35, Oligo 50 and Oligo 70 at an electric field strength of 280 V/cm that were separated in NIM-PMMA-COC8007 device. (B) Histogram of effective migration time of Oligo35, Oligo50 and Oligo70 at a field strength of 280 V/cm that were separated in IM-PMMA-COC 8007 device. Histograms were fitted in to a Gaussian distribution and each bin represent 0.05 s for n = 100 events. (C) Calculated separation resolution between the 3 ssDNA. (D) Contact angle of IM-PMMA and NIM-PMMA before and after oxygen plasma modification.

Figure 7.

(A) Histogram of migration time for ssDNAs at an effective column length of 30 μm through a 100 nm × 100 nm NIM-PMMA nanochannel at a field strength of 280 V/cm. Histograms were fit to a Gaussian and each histogram represents 100 events. (B) Comparison of peak resolution at an effective column length of 60 μm and 30 μm. (C) Calculated theoretical plates for ssDNAs at effective column lengths of 30 μm and 60 μm. N = 5.54 (t_m/w_0.5)² was used to calculate the number of theoretical plates and $\frac{N\times 100}{\mathit{\text{column length }}(\mathit{\text{cm}})}$ to obtained theoretical plates in units of m⁻¹.