Quantification of Low Concentrations of DNA Using Single Molecule Detection and Velocity Measurement in a Microchannel |
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Authors: | Shu-Yi Chao Yi-Ping Ho Vasudev J Bailey Tza-Huei Wang |
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Institution: | (1) Department of Mechanical Engineering, The Johns Hopkins University, Baltimore, MD 21218, USA;(2) Department of Biomedical Engineering, The Johns Hopkins University, Baltimore, MD 21218, USA;(3) Whitaker Biomedical Engineering Institute, The Johns Hopkins University, Baltimore, MD 21218, USA;(4) The Johns Hopkins School of Medicine, The Johns Hopkins University, Baltimore, MD 21218, USA |
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Abstract: | We present a novel method for quantifying low concentrations of DNA based on single molecule detection (SMD) for molecular
counting and flow measurements inside a microchannel. A custom confocal fluorescence spectroscopic system is implemented to
detect fluorescent bursts emitted from stained DNA molecules. Measurements are made one molecule at a time as they flow through
a femtoliter-sized laser focal probe. Durations of single molecule fluorescent bursts, which are found to be strongly related
to the molecular transit times through the detection region, are statistically analyzed to determine the in situ flow speed
and subsequently the sample volume flowing through the focal probe. Therefore, the absolute concentration of a DNA sample
can be quantified based on the single molecule fluorescent counts from the DNA molecules and the associated probe volume for
a measured time course. To validate this method for quantifying low concentrations of biomolecules, we tested samples of pBR322
DNA ranging from 1 pM to 10 fM (∼3 ng/ml to 30 pg/ml). Besides molecular quantification, we also demonstrate this method to
be a precise and non-invasive way for flow profiling within a microchannel. |
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Keywords: | Single molecule detection DNA quantification Flow profiling Microchannel |
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