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

Lisa-Maria Needham, Carlos Saavedra, Julia K. Rasch, Daniel Sole-Barber, Beau S. Schweitzer, Alex J. Fairhall, Cecilia H. Vollbrecht, Sushu Wan, Yulia Podorova, Anders J. Bergsten, Brandon Mehlenbacher, Zhao Zhang, Lukas Tenbrake, Jovanna Saimi, Lucy C. Kneely, Jackson S. Kirkwood, Hannes Pfeifer, Edwin R. Chapman and Randall H. Goldsmith ()
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Lisa-Maria Needham: University of Wisconsin–Madison
Carlos Saavedra: University of Wisconsin–Madison
Julia K. Rasch: University of Wisconsin–Madison
Daniel Sole-Barber: University of Wisconsin–Madison
Beau S. Schweitzer: University of Wisconsin–Madison
Alex J. Fairhall: University of Wisconsin–Madison
Cecilia H. Vollbrecht: University of Wisconsin–Madison
Sushu Wan: University of Wisconsin–Madison
Yulia Podorova: University of Wisconsin–Madison
Anders J. Bergsten: University of Wisconsin–Madison
Brandon Mehlenbacher: University of Wisconsin–Madison
Zhao Zhang: University of Wisconsin–Madison
Lukas Tenbrake: Universität Bonn
Jovanna Saimi: University of Wisconsin–Madison
Lucy C. Kneely: University of Wisconsin–Madison
Jackson S. Kirkwood: University of Wisconsin–Madison
Hannes Pfeifer: Universität Bonn
Edwin R. Chapman: University of Wisconsin–Madison
Randall H. Goldsmith: University of Wisconsin–Madison

Nature, 2024, vol. 629, issue 8014, 1062-1068

Abstract: Abstract Most chemistry and biology occurs in solution, in which conformational dynamics and complexation underlie behaviour and function. Single-molecule techniques1 are uniquely suited to resolving molecular diversity and new label-free approaches are reshaping the power of single-molecule measurements. A label-free single-molecule method2–16 capable of revealing details of molecular conformation in solution17,18 would allow a new microscopic perspective of unprecedented detail. Here we use the enhanced light–molecule interactions in high-finesse fibre-based Fabry–Pérot microcavities19–21 to detect individual biomolecules as small as 1.2 kDa, a ten-amino-acid peptide, with signal-to-noise ratios (SNRs) >100, even as the molecules are unlabelled and freely diffusing in solution. Our method delivers 2D intensity and temporal profiles, enabling the distinction of subpopulations in mixed samples. Notably, we observe a linear relationship between passage time and molecular radius, unlocking the potential to gather crucial information about diffusion and solution-phase conformation. Furthermore, mixtures of biomolecule isomers of the same molecular weight and composition but different conformation can also be resolved. Detection is based on the creation of a new molecular velocity filter window and a dynamic thermal priming mechanism that make use of the interplay between optical and thermal dynamics22,23 and Pound–Drever–Hall (PDH) cavity locking24 to reveal molecular motion even while suppressing environmental noise. New in vitro ways of revealing molecular conformation, diversity and dynamics can find broad potential for applications in the life and chemical sciences.

Date: 2024
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DOI: 10.1038/s41586-024-07370-8

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