Free-solution oligonucleotide separation in nanoscale channels

In this paper, we report an experimental study of electrokinetic transport and separation of double-stranded deoxyribonucleic acid (dsDNA) oligonucleotides in custom-fabricated fused-silica nanochannels filled with a gel-free sodium borate aqueous buffer. Mixtures of fluorescently labeled dsDNA molecules in the range of 10-100 base pair (bp), fluorescein, and fluorescein-12-UTP (UTP) were separated in less than 120 s in channels of depth ranging from 40 to 1560 nm. We varied the channel depth and background buffer concentration to achieve a 0.006-0.2 range of Debye length-to-channel-half-depth ratio lambda(D)/h), and a 0.004-1.7 range of the ratio of length of dsDNA molecule to channel half-depth (l/h). We find observed oligonucleotide migration times depend on both l/h and lambda(D)/h. Electrophoretic mobility estimates agree well with published (micrometer-scale channel) values for background electrolyte (BGE) concentrations greater than approximately 10 mM. At BGE concentrations of 1 and 5 mM, mobility estimates in our nanochannels are higher than published values. Of the cases studied, the highest separation sensitivities were achieved in 100 nm channels with 1-10 mM ion density buffers. Potential applications of this technology include rapid small-scale sequencing and other fluorescence-based oligonucleotide separation and detection assays: