Single molecule fluorescence under conditions of fast flow

Area of Science:

• Analytical Chemistry
• Physical Chemistry
• Biophysics

Background:

• Confocal fluorescence spectroscopy is crucial for single-molecule analysis.
• Characterizing and counting single molecules often faces limitations due to photophysical processes and short residence times in the detection volume.

Purpose of the Study:

• To experimentally determine optimal flow velocities for single-molecule characterization and counting using microfluidics.
• To enhance data acquisition rates for two-color coincidence detection (TCCD) and single pair Förster resonance energy transfer (spFRET).

Main Methods:

• Utilized a microfluidic device for two-color coincidence detection (TCCD) and single pair Förster resonance energy transfer (spFRET).
• Employed confocal fluorescence spectroscopy to analyze molecules flowing at speeds up to 10 cm s(-1).
• Investigated the impact of flow velocities (≥0.5 cm s(-1)) on reducing competing photophysical processes and compensating for short residence times (10-200 μs).

Main Results:

• Achieved significant increases in data acquisition rates: up to 38-fold for TCCD at 5 cm s(-1) and 18-fold for spFRET at 2 cm s(-1) compared to diffusion.
• Demonstrated enhanced event rates for structural characterization at slower speeds (2 cm s(-1) for TCCD, 1 cm s(-1) for spFRET), with 33-fold and 16-fold increases, respectively.
• Enabled rapid quantification of analytes over 4 orders of magnitude, detecting 50 fM dual-labeled sample in ~8 s using TCCD at 5 cm s(-1).

Conclusions:

• Optimized flow velocities in microfluidic systems significantly boost the efficiency of single-molecule detection techniques like TCCD and spFRET.
• High-throughput single-molecule counting and characterization are achievable, enabling rapid quantification of analytes with high statistical confidence.
• This approach offers a powerful method for sensitive and rapid analysis of molecular samples.