Label-free detection and profiling of individual solution-phase molecules

Area of Science:

• Biophysics
• Analytical Chemistry
• Physical Chemistry

Background:

• Chemistry and biology in solution rely on conformational dynamics and complexation.
• Single-molecule techniques are vital for resolving molecular diversity.
• Label-free methods enhance single-molecule measurement capabilities.

Purpose of the Study:

• Develop a label-free single-molecule method to reveal molecular conformation in solution.
• Provide a new microscopic perspective with unprecedented detail.
• Enable detailed analysis of biomolecular behavior and function.

Main Methods:

• Utilized enhanced light-molecule interactions in high-finesse fiber-based Fabry-Pérot microcavities.
• Employed a new molecular velocity filter window and dynamic thermal priming mechanism.
• Leveraged Pound-Drever-Hall (PDH) cavity locking to suppress environmental noise.

Main Results:

• Detected individual, unlabelled biomolecules as small as 1.2 kDa (ten-amino-acid peptide) with signal-to-noise ratios >100.
• Obtained 2D intensity and temporal profiles, distinguishing subpopulations in mixed samples.
• Observed a linear relationship between passage time and molecular radius, enabling conformation and diffusion analysis.
• Resolved mixtures of biomolecule isomers with identical weight and composition but different conformations.

Conclusions:

• The developed method offers a powerful new tool for analyzing molecular conformation, diversity, and dynamics in solution.
• This technique provides crucial information about diffusion and solution-phase conformation.
• Potential broad applications exist in the life and chemical sciences for in vitro analysis.