Single-molecule analysis enables free solution hydrodynamic separation using yoctomole levels of DNA.

Single-molecule free solution hydrodynamic separation (SML-FSHS) cohesively integrates cylindrical illumination confocal spectroscopy with free solution hydrodynamic separation. This technique enables single-molecule analysis of size separated DNA with 100% mass detection efficiency, high sizing resolution and wide dynamic range, surpassing the performance of single molecule capillary electrophoresis. Furthermore, SML-FSHS required only a bare fused silica microcapillary and simple pressure control rather than complex high voltage power supplies, sieving matrices, and wall coatings. The wide dynamic range and high sizing resolution of SML-FSHS was demonstrated by separating both large DNA (23 vs 27 kbp) and small DNA (100 vs 200 bp) under identical conditions. Separations were successfully performed with near zero sample consumption using as little as 5 pL of sample and 240 yoctomoles (∼150 molecules) of DNA. Quantitative accuracy was predominantly limited by molecular shot noise. Furthermore, the ability of this method to analyze of single molecule nanosensors was investigated. SML-FSHS was used to examine the thermodynamic equilibrium between stochastically open molecular beacon and target-bound molecular beacon in the detection of E. coli 16s rRNA targets.

[1]  Tza-Huei Wang,et al.  Decoding circulating nucleic acids in human serum using microfluidic single molecule spectroscopy. , 2010, Journal of the American Chemical Society.

[2]  R. Mathies,et al.  Single-molecule detection of DNA separations in microfabricated capillary electrophoresis chips employing focused molecular streams. , 1999, Analytical chemistry.

[3]  Norman J. Dovichi,et al.  Single-Molecule Detection in Capillary Electrophoresis: Molecular Shot Noise as a Fundamental Limit to Chemical Analysis , 1996 .

[4]  Gang Bao,et al.  Dual FRET molecular beacons for mRNA detection in living cells. , 2004, Nucleic acids research.

[5]  M. Finney.  Pulsed-field gel electrophoresis. , 2001, Current protocols in molecular biology.

[6]  Weihong Tan,et al.  Locked nucleic acid molecular beacons. , 2005, Journal of the American Chemical Society.

[7]  Yongseong Kim,et al.  Electrostatic and Hydrodynamic Separation of DNA Fragments in Capillary Tubes , 1996 .

[8]  R. Allen,et al.  Free solution hydrodynamic separation of DNA fragments from 75 to 106,000 base pairs in a single run. , 2010, Journal of the American Chemical Society.

[9]  B G Johansson,et al.  Agarose gel electrophoresis. , 1972, Scandinavian journal of clinical and laboratory investigation. Supplementum.

[10]  E. Arriaga,et al.  Capillary electrophoresis in bioanalysis. , 2008, Analytical chemistry.

[11]  S. Boppart,et al.  Use of DNA and Peptide Nucleic Acid Molecular Beacons for Detection and Quantification of rRNA in Solution and in Whole Cells , 2003, Applied and Environmental Microbiology.

[12]  Tza-Huei Wang,et al.  Cylindrical illumination confocal spectroscopy: rectifying the limitations of confocal single molecule spectroscopy through one-dimensional beam shaping. , 2008, Biophysical journal.

[13]  Alexander N. Glazer,et al.  Stable dye–DNA intercalation complexes as reagents for high-sensitivity fluorescence detection , 1992, Nature.

[14]  C. Effenhauser,et al.  Integrated capillary electrophoresis on flexible silicone microdevices:  analysis of DNA restriction fragments and detection of single DNA molecules on microchips. , 1997, Analytical chemistry.

[15]  H. Yeh,et al.  Single-quantum-dot-based DNA nanosensor , 2005, Nature materials.

[16]  M. Morris,et al.  Separation of nucleic acids by capillary electrophoresis in cellulose solutions with mono- and bis-intercalating dyes. , 1994, Analytical chemistry.

[17]  R. Allen,et al.  Bare nanocapillary for DNA separation and genotyping analysis in gel-free solutions without application of external electric field. , 2008, Analytical chemistry.

[18]  R. Mathies,et al.  Single molecule fluorescence burst detection of DNA fragments separated by capillary electrophoresis. , 1995, Analytical chemistry.

[19]  R. Tijssen,et al.  Hydrodynamic chromatography of macromolecules in open microcapillary tubes , 1986 .

[20]  C. Dekker,et al.  Pressure-driven transport of confined DNA polymers in fluidic channels , 2006, Proceedings of the National Academy of Sciences.