Skip to main content
SpringerLink
Log in

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

  • Original Paper
  • Published:
Journal of Fluorescence Aims and scope Submit manuscript

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  1. Belkum AV (2003) Molecular diagnostics in medical microbiology: yesterday, today and tomorrow. Curr Opin Pharmacol 3:497–501

    Article  PubMed  CAS  Google Scholar 

  2. Gingeras TR, Higuchi R, Kricka LJ, Lo YMD, Wittwer CT (2005) Fifty years of molecular (DNA/RNA) diagnostics. Clin Chem 51:661–667

    Article  PubMed  CAS  Google Scholar 

  3. Halford WP, Falco VC, Gebhardt BM, Carr DJJ (1999) The inherent quantitative capacity of the reverse transcription polymerase chain reaction. Anal Biochem 266:181–191

    Article  PubMed  CAS  Google Scholar 

  4. Pollack JR, Perou CM, Alizadeh AA, Eisen MB, Pergamenschikov A, Williams CF, Jeffrey SS, Botstein D, Brown PO (1999) Genome-wide analysis of DNA copy-number changes using cDNA microarrays. Nat Genet 23:41–46

    Article  PubMed  CAS  Google Scholar 

  5. Kwok PY (2001) Methods for genotyping single nucleotide polymorphisms. Ann Rev Genomics Hum Genet 2:235–258

    Article  CAS  Google Scholar 

  6. Beaudet L, Bedard J, Breton B, Mercuri RJ, Budarf ML (2001) Homogeneous assays for single-nucleotide polymorphism typing using AlphaScreen. Genome Res 11(4):600–608

    Article  PubMed  CAS  Google Scholar 

  7. Myakishev MV, Khripin Y, Hu S, Hamer DH (2001) High-throughput SNP genotyping by allele-specific PCR with universal energy-transfer-labeled primers. Genome Res 11(1):163–169

    Article  PubMed  CAS  Google Scholar 

  8. Herman JG, Baylin SB (2003) Mechanisms of disease: gene silencing in cancer in association with promoter hypermethylation. N Engl J Med 349(21):2042–2054

    Article  PubMed  CAS  Google Scholar 

  9. Peccoud J, Jacob C (1996) Theoretical uncertainty of measurements using quantitative polymerase chain reaction. Biophys J 71:101–108

    Article  PubMed  CAS  Google Scholar 

  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 4:684–691

    Google Scholar 

  11. Knemeyer JP, Marmé N, Sauer M, (2000) Probes for detection of specific DNA sequences at the single-molecule level. Anal Chem 72:3717–3724

    Article  PubMed  CAS  Google Scholar 

  12. Zhang CY, Yeh HC, Kuroki MT, Wang TH (2005) Single-quantum-dot-based DNA nanosensor. Nat Mater 4:826–831

    Article  PubMed  Google Scholar 

  13. Yeh HC, Ho YP, Shih IM, Wang TH (2006) Homogenous point mutation detection by quantum dot-mediated two-color fluorescence coincidence analysis. Nucleic Acids Res 34:e35

    Article  PubMed  CAS  Google Scholar 

  14. Li HT, Ying LM, Green JJ, Balasubramanian S, Klenerman D (2003) Ultrasensitive coincidence fluorescence detection of single DNA molecules. Anal Chem 751:664–1670

    Google Scholar 

  15. Wang TH, Peng YH, Zhang CY, Wong PK, Ho CM (2005) Single-molecule tracing on a fluidic microchip for quantitative detection of low-abundance nucleic acids. J Am Chem Soc 127:5354–5359

    Article  PubMed  CAS  Google Scholar 

  16. Castro A, Williams JGK (1997) Single-molecule detection of specific nucleic acid sequences in unamplified genomic DNA. Anal Chem 69:3915–3920

    Article  PubMed  CAS  Google Scholar 

  17. Zhang CY, Chao SY, Wang TH (2005) Comparative quantification of nucleic acids using single-molecule detection and molecular beacons. Analyst 130(4):483–488

    Article  PubMed  CAS  Google Scholar 

  18. Neely LA, Patel S, Garver J, Gallo M, Hackett M, McLaughlin S, Nadel M, Harris J, Gullans S, Rooke J (2006) A single-molecule method for the quantitation of microRNA gene expression. Nat Methods 3:41–46

    Article  PubMed  CAS  Google Scholar 

  19. Devasenathipathy S, Santiago JG, Wereley ST, Meinhart CD, Takehara K (2003) Particle imaging techniques for microfabricated fluidic systems. Exp Fluids 34:504–514

    Google Scholar 

  20. Santiago JG, Wereley ST, Meinhart CD, Beebe DJ, Adrian RJ (1998) A particle image velocimetry system for microfluidics. Exp Fluids 25:316–319

    Article  CAS  Google Scholar 

  21. Wang W, Liu Y, Sonek GJ, Berns MW, Keller RA (1995) Optical trapping and fluorescence detection in laminar flow streams. Appl Phys Lett 67:1057–1059

    Article  CAS  Google Scholar 

  22. Gosch M, Blom H, Holm J, Heino T, Rigler R (2000) Hydrodynamic flow profiling in microchannel structures by single molecule fluorescence correlation spectroscopy. Anal Chem 72:3260–3265

    Article  PubMed  CAS  Google Scholar 

  23. Kunst BH, Schots A, Visser A (2002) Detection of flowing fluorescent particles in a microcapillary using fluorescence correlation spectroscopy. Anal Chem 74:5350–5357

    Article  PubMed  CAS  Google Scholar 

  24. Hsu TR (2001) MEMS and microsystems: design and manufacture. McGraw-Hill

  25. Watson N (1988) A new revision of the sequence of plasmid pBR322. Gene 70:399–403

    Article  PubMed  CAS  Google Scholar 

  26. Haugland RP (2002) Handbook of fluorescent probes and research products. Molecular Probes, Eugene

    Google Scholar 

  27. Fox RW, Mc Donald AT, Pritchard PJ (2003) Introduction to fluid mechanics 6 ed. Wiley, New York

    Google Scholar 

Download references

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.

Author information

Authors and Affiliations

  1. Department of Mechanical Engineering, The Johns Hopkins University, Baltimore, MD, 21218, USA

    Shu-Yi Chao, Yi-Ping Ho & Tza-Huei Wang

  2. Department of Biomedical Engineering, The Johns Hopkins University, Baltimore, MD, 21218, USA

    Vasudev J. Bailey & Tza-Huei Wang

  3. Whitaker Biomedical Engineering Institute, The Johns Hopkins University, Baltimore, MD, 21218, USA

    Tza-Huei Wang

  4. The Johns Hopkins School of Medicine, The Johns Hopkins University, Baltimore, MD, 21218, USA

    Vasudev J. Bailey

Authors
  1. Shu-Yi Chao
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yi-Ping Ho
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Vasudev J. Bailey
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Tza-Huei Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Tza-Huei Wang.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Chao, SY., Ho, YP., Bailey, V.J. et al. Quantification of Low Concentrations of DNA Using Single Molecule Detection and Velocity Measurement in a Microchannel. J Fluoresc 17, 767–774 (2007). https://doi.org/10.1007/s10895-007-0194-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10895-007-0194-0

Keywords

Search

Navigation