DNA microelectrophoresis using double focus fluorescence correlation spectroscopy.

Area of Science:

• Biophysics
• Analytical Chemistry
• Microfluidics

Background:

• Accurate measurement of DNA fragment electrophoretic mobility is crucial for microfluidic analysis.
• Understanding electrokinetic flow profiles in microchannels is essential for separation techniques.

Purpose of the Study:

• To determine electrophoretic mobilities and diffusion constants of short double-stranded DNA (dsDNA) fragments using dfFCS.
• To investigate the influence of DNA length and polymer networks on electrophoretic behavior.
• To assess the potential of dfFCS for high-resolution, real-time micro-analysis.

Main Methods:

• Utilized double focus fluorescence correlation spectroscopy (dfFCS) for high-resolution measurements.
• Analyzed electrokinetic flow profiles in microfluidic channels with 1 µm spatial resolution.
• Separated electroosmotic and electrophoretic contributions to the flow.

Main Results:

• Free solution mobility of dsDNA fragments was independent of length (75-1019 bp).
• Diffusion constants followed a length-dependent rod-diffusion model.
• Electrophoretic velocities became size-dependent (power-law exponent 0.28-0.31) in a 3% PEO network, explained by Manning condensation and hydrodynamic retardation.
• Distinguishable peaks were observed for mixtures of dsDNA fragments in dfFCS cross-correlation functions.

Conclusions:

• dfFCS provides high spatial resolution and speed for real-time micro-analysis of DNA fragments.
• Electrophoretic mobility is length-independent in free solution but exhibits size-dependence in polymer networks.
• Modified Nernst Einstein relation effectively models electrophoretic mobilities considering condensation and retardation effects.