An analytical system based on a compact flow cytometer for DNA fragment sizing and single-molecule detection

Area of Science:

• Biotechnology
• Analytical Chemistry
• Molecular Biology

Background:

• Previous flow systems accurately sized linear DNA fragments (564 bp to 4 x 10^5 bp).
• B-phycoerythrin (B-PE) was the first fluorophore detected at the single-molecule level in flow.

Purpose of the Study:

• To develop a compact, low-cost, high-sensitivity flow system for biomolecule analysis.
• To demonstrate accurate DNA fragment sizing and single-molecule detection using enhanced photon-counting technology.

Main Methods:

• Utilized a simplified, compact flow system with a solid-state laser and single-photon avalanche photodiode detector (SSAPD).
• Developed specialized software for processing photon-counting data to extract molecular features.
• Analyzed SYTOX-orange-stained DNA fragments and B-phycoerythrin solutions.

Main Results:

• Achieved DNA fragment sizing for fragments from 125 bp to 5 x 10^5 bp with high accuracy (CV of 1.2% for 48.5 kbp lambda DNA).
• Demonstrated baseline resolution of individual B-PE molecules with low laser power (0.5 mW) and high signal-to-noise ratio (SNR ≈ 30).
• Detected approximately 100 photons per molecule, indicating high sensitivity.

Conclusions:

• Developed a compact, low-power, high-sensitivity system for detecting small DNA fragments (125 bp) and individual B-PE molecules.
• Optimal laser power (<1.0 mW) allows for system miniaturization and cost reduction.
• Analytical software enables detailed analysis of biomolecules, extending capabilities beyond conventional flow cytometry.