Journal of Fluorescence

, Volume 17, Issue 6, pp 767–774 | Cite as

Quantification of Low Concentrations of DNA Using Single Molecule Detection and Velocity Measurement in a Microchannel

  • Shu-Yi Chao
  • Yi-Ping Ho
  • Vasudev J. Bailey
  • Tza-Huei Wang
Original Paper

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.

Keywords

Single molecule detection DNA quantification Flow profiling Microchannel 

Notes

Acknowledgements

This work is supported by NSF and DARPA. We thank the members of the BioMEMS and Single Molecule Dynamics lab for the stimulating discussion and their invaluable help.

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Copyright information

© Springer Science+Business Media, LLC 2007

Authors and Affiliations

  • Shu-Yi Chao
    • 1
  • Yi-Ping Ho
    • 1
  • Vasudev J. Bailey
    • 2
    • 4
  • Tza-Huei Wang
    • 1
    • 2
    • 3
  1. 1.Department of Mechanical EngineeringThe Johns Hopkins UniversityBaltimoreUSA
  2. 2.Department of Biomedical EngineeringThe Johns Hopkins UniversityBaltimoreUSA
  3. 3.Whitaker Biomedical Engineering InstituteThe Johns Hopkins UniversityBaltimoreUSA
  4. 4.The Johns Hopkins School of MedicineThe Johns Hopkins UniversityBaltimoreUSA

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