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 (lambdaD/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 lambdaD/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.
Pennathur
Sumita
S
Mechanical Engineering Department, Stanford University, Stanford, California 94305, USA.
Baldessari
Fabio
F
Santiago
Juan G
JG
Kattah
Michael G
MG
Steinman
Jonathan B
JB
Utz
Paul J
PJ
D001881:Borates / Q000737:chemistry; D004247:DNA / Q000032:analysis*; D044085:Microfluidics / Q000295:instrumentation / Q000379:methods; D036103:Nanotechnology / Q000295:instrumentation / Q000379:methods; D009841:Oligonucleotides / Q000032:analysis*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012822:Silicon Dioxide / Q000737:chemistry*; D013997:Time Factors; D014867:Water / Q000737:chemistry
D001881:Borates; D009841:Oligonucleotides; D014867:Water; D012822:Silicon Dioxide; D004247:DNA; C010634:sodium borate