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Rapid quantitative analysis using a single molecule counting approach.

Christina M D'Antoni1, Martin Fuchs, John L Harris

  • 1U.S. Genomics, 12 Gill St., Suite 4700, Woburn, MA 01801, USA.

Analytical Biochemistry
|March 22, 2006
PubMed
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This study introduces a new method for counting dual-labeled molecules using fluorescent probes, enabling sensitive detection of biomolecules. The approach accurately quantifies targets even at subpicomolar concentrations in complex samples.

Area of Science:

  • Biotechnology
  • Analytical Chemistry
  • Molecular Biology

Background:

  • Single molecule detection offers high sensitivity for biomolecule quantitation using paired fluorescent probes.
  • Analytical capabilities and data analysis of such dual-probe platforms remain underexplored.
  • Accurate quantitation requires robust methods to distinguish bound probes from unbound ones.

Purpose of the Study:

  • To develop and evaluate a novel data analysis methodology for single molecule detection using paired fluorescent probes.
  • To assess the analytical performance of a rapid, automated, multicolor single molecule detection apparatus.
  • To demonstrate the platform's capability for sensitive quantitation of biomolecules in complex backgrounds.

Main Methods:

  • Developed a novel extension of coincident event counting for analyzing fluorescent probe signals.

Related Experiment Videos

  • Corrected for unbound probes passing simultaneously through the interrogation zone.
  • Evaluated the method on three fluorescence channel combinations and compared it to spatial cross-correlation.

    • Main Results:

    • The coincident event counting approach demonstrated a wider linear dynamic response compared to spatial cross-correlation.
    • Successfully detected subpicomolar concentrations of a model RNA target in a complex RNA background.
    • The apparatus is rapid, automated, and sensitive, utilizing multicolor detection.

    Conclusions:

    • The developed fluorescent event counting approach provides a general and sensitive tool for quantitative analysis.
    • This method is applicable to various analytes, including nucleic acids and proteins, with specific probe pairs.
    • The platform enables rapid and sensitive quantification of biomolecules at the single-molecule level.