Quantification of Low Concentrations of DNA Using Single Molecule Detection and Velocity Measurement in a Microchannel
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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.
KeywordsSingle molecule detection DNA quantification Flow profiling Microchannel
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.
- 10.Barnard FVR, Pecheniuk N, Slattery M, Walsh T (1998) PCR bias toward the wild-type k-ras and p53 sequences: implications for PCR detection of mutations and cancer diagnosis. Biotechniques 4:684–691Google Scholar
- 14.Li HT, Ying LM, Green JJ, Balasubramanian S, Klenerman D (2003) Ultrasensitive coincidence fluorescence detection of single DNA molecules. Anal Chem 751:664–1670Google Scholar
- 19.Devasenathipathy S, Santiago JG, Wereley ST, Meinhart CD, Takehara K (2003) Particle imaging techniques for microfabricated fluidic systems. Exp Fluids 34:504–514Google Scholar
- 24.Hsu TR (2001) MEMS and microsystems: design and manufacture. McGraw-HillGoogle Scholar
- 26.Haugland RP (2002) Handbook of fluorescent probes and research products. Molecular Probes, EugeneGoogle Scholar
- 27.Fox RW, Mc Donald AT, Pritchard PJ (2003) Introduction to fluid mechanics 6 ed. Wiley, New YorkGoogle Scholar