Rapid quantitative analysis using a single molecule counting approach

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.
• 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.