We report a microfluidic platform for sizing and separation of DNA called PicoSep that is among the most sensitive to date, requiring only yoctomoles of DNA (10−24 moles) and picoliters of sample (<10 pL). The cohesive integration of cylindrical illumination confocal spectroscopy based (CICS) single molecule counting with free solution hydrodynamic separation increases detection sensitivity >103-fold over traditional laser induced fluorescence (LIF) and vastly improves quantification accuracy. Separation is performed via single molecule free solution hydrodynamic separation (SML-FSHS). SML-FSHS relies on the wall exclusion mechanism to separate DNA as it is driven down buffer filled microchannel. High separation efficiency (plate number = 105 – 106) and high sizing resolution (37 bp – 2.1 kbp) are obtained across a wide dynamic range of DNA (100 bp – 27 kbp) in a single separation. Quantitative accuracy up to the limits imposed by molecular shot noise is achieved, and a limit of detection <20 molecules is demonstrated. Through the development of PicoSep, we have significantly reduced the cost and complexity of single molecule instrumentation while achieving sizing and quantification performance that surpasses capillary electrophoresis (CE). Unlike CE, no viscous sieving matrices, high voltage power supplies, or capillary wall coatings are necessary, making devices inexpensive, simple to fabricate, and easy to incorporate into lab on a chip systems.
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ASME 2012 10th International Conference on Nanochannels, Microchannels, and Minichannels collocated with the ASME 2012 Heat Transfer Summer Conference and the ASME 2012 Fluids Engineering Division Summer Meeting
July 8–12, 2012
Rio Grande, Puerto Rico, USA
Conference Sponsors:
- Heat Transfer Division
- Fluids Engineering Division
ISBN:
978-0-7918-4479-3
PROCEEDINGS PAPER
An Integrated Platform for Single Molecule Free Solution Hydrodynamic Separation Using Yoctomoles of DNA and Picoliter Samples
Kelvin J. Liu,
Kelvin J. Liu
Johns Hopkins University, Baltimore, MD
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Tushar D. Rane,
Tushar D. Rane
Johns Hopkins University, Baltimore, MD
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Yi Zhang,
Yi Zhang
Johns Hopkins University, Baltimore, MD
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Cyrus W. Beh,
Cyrus W. Beh
Johns Hopkins University, Baltimore, MD
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Sarah M. Friedrich,
Sarah M. Friedrich
Johns Hopkins University, Baltimore, MD
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Dong Jin Shin,
Dong Jin Shin
Johns Hopkins University, Baltimore, MD
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Tza-Huei Wang
Tza-Huei Wang
Johns Hopkins University, Baltimore, MD
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Kelvin J. Liu
Johns Hopkins University, Baltimore, MD
Tushar D. Rane
Johns Hopkins University, Baltimore, MD
Yi Zhang
Johns Hopkins University, Baltimore, MD
Cyrus W. Beh
Johns Hopkins University, Baltimore, MD
Sarah M. Friedrich
Johns Hopkins University, Baltimore, MD
Dong Jin Shin
Johns Hopkins University, Baltimore, MD
Tza-Huei Wang
Johns Hopkins University, Baltimore, MD
Paper No:
ICNMM2012-73154, pp. 1-5; 5 pages
Published Online:
July 22, 2013
Citation
Liu, KJ, Rane, TD, Zhang, Y, Beh, CW, Friedrich, SM, Shin, DJ, & Wang, T. "An Integrated Platform for Single Molecule Free Solution Hydrodynamic Separation Using Yoctomoles of DNA and Picoliter Samples." Proceedings of the ASME 2012 10th International Conference on Nanochannels, Microchannels, and Minichannels collocated with the ASME 2012 Heat Transfer Summer Conference and the ASME 2012 Fluids Engineering Division Summer Meeting. ASME 2012 10th International Conference on Nanochannels, Microchannels, and Minichannels. Rio Grande, Puerto Rico, USA. July 8–12, 2012. pp. 1-5. ASME. https://doi.org/10.1115/ICNMM2012-73154
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