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Free-solution oligonucleotide separation in nanoscale channels.

Sumita Pennathur1, Fabio Baldessari, Juan G Santiago

  • 1Mechanical Engineering Department, Stanford University, Stanford, California 94305, USA.

Analytical Chemistry
|September 22, 2007
PubMed
Summary

This study demonstrates rapid separation of double-stranded deoxyribonucleic acid (dsDNA) in nanochannels using electrokinetic transport. Optimized conditions achieved high sensitivity for oligonucleotide separation and detection assays.

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Area of Science:

  • Nanotechnology
  • Biophysics
  • Analytical Chemistry

Background:

  • Electrokinetic phenomena are crucial for microfluidic separations.
  • Separating double-stranded deoxyribonucleic acid (dsDNA) oligonucleotides presents challenges in micro- and nanochannels.
  • Understanding the influence of channel dimensions and buffer concentration on dsDNA transport is essential.

Purpose of the Study:

  • To experimentally investigate electrokinetic transport and separation of dsDNA oligonucleotides in nanochannels.
  • To determine the impact of channel depth and buffer concentration on separation efficiency.
  • To explore potential applications in rapid oligonucleotide analysis.

Main Methods:

  • Fabrication of custom fused-silica nanochannels with depths from 40 to 1560 nm.
  • Utilizing a gel-free sodium borate aqueous buffer.
  • Separating fluorescently labeled dsDNA (10-100 bp), fluorescein, and fluorescein-12-UTP (UTP) using electrokinetic transport.
  • Varying Debye length-to-channel-half-depth (λD/h) and dsDNA length-to-channel-half-depth (l/h) ratios.

Main Results:

  • Oligonucleotide separation achieved in under 120 seconds.
  • Migration times were found to depend on both l/h and λD/h ratios.
  • Electrophoretic mobility matched published data at background electrolyte concentrations >10 mM.
  • Higher mobility estimates than published values were observed at 1-5 mM concentrations.
  • Optimal separation sensitivity was achieved in 100 nm channels with 1-10 mM ion density buffers.

Conclusions:

  • Electrokinetic transport in nanochannels enables efficient dsDNA oligonucleotide separation.
  • Channel geometry and buffer conditions significantly influence separation dynamics.
  • This technique shows promise for rapid, small-scale sequencing and fluorescence-based oligonucleotide assays.