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

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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 method2,3,4,5,6,7,8,9,10,11,12,13,14,15,16 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 microcavities19,20,21 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.

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Fig. 1: Measurement apparatus and resonance scan.
Fig. 2: Signals from individual molecules.
Fig. 3: Diffusion information from single-molecule profiles.
Fig. 4: Resolution of mixtures.
Fig. 5: Mechanism of cavity-enhanced single-molecule detection.

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Data availability

A representative sample of the raw data supporting the findings of this work is openly available on figshare at https://figshare.com/articles/dataset/Label-free_detection_and_profiling_of_individual_solution-phase_molecules-_sample_raw_data/25463965, under the title ‘Label-free detection and profiling of individual solution-phase molecules- sample raw data’, with the following ref. 64. The data are associated with Figs. 2 and 3. The sample data are in .csv format and available without restrictions.

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Acknowledgements

This work was mainly financed by the National Institutes of Health (NIH, R01GM136981), with resonator construction supported by the Q-NEXT Quantum Center, a U.S. Department of Energy (DOE), Office of Science, National Quantum Information Science Research Centers, under award number DE-FOA-0002253. Further instrumentation development was supported by the Center for Molecular Quantum Transduction, an Energy Frontier Research Center financed by DOE, Office of Science, BES under award DE-SC0021314, the National Science Foundation (NSF) Quantum Leap Challenge Institute for Hybrid Quantum Architectures and Networks, award no. 2016136, and by Schmidt Futures. L.-M.N. was partially financed by the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement no. 886216. E.R.C. was financed by the NIH (MH061876 and NS097362). H.P. was financed by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany’s Excellence Strategy – Cluster of Excellence Matter and Light for Quantum Computing (ML4Q) EXC 2004/1 – 390534769. We thank D. Hunger, T. Northup and S. Vanga for help in initial fibre mirror and microcavity fabrication, X. Huang and B. Cullinane for conversations on diffusion, M. Saffman for early conversations on fibre microcavities and B. Thompson and T. Drier for instrumentation development.

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Author notes

  1. Lisa-Maria Needham

    Present address: School of the Biological Sciences, University of Cambridge, Cambridge, UK

  2. Cecilia H. Vollbrecht

    Present address: Department of Chemistry and Biochemistry, Kalamazoo College, Kalamazoo, MI, USA

Authors and Affiliations

  1. Department of Chemistry, University of Wisconsin–Madison, Madison, WI, USA

    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, Jovanna Saimi, Lucy C. Kneely, Jackson S. Kirkwood & Randall H. Goldsmith

  2. Yusuf Hamied Department of Chemistry, University of Cambridge, Cambridge, UK

    Lisa-Maria Needham

  3. Howard Hughes Medical Institute, University of Wisconsin–Madison, Madison, WI, USA

    Zhao Zhang & Edwin R. Chapman

  4. Department of Neuroscience, University of Wisconsin–Madison, Madison, WI, USA

    Zhao Zhang & Edwin R. Chapman

  5. Institut für Angewandte Physik, Universität Bonn, Bonn, Germany

    Lukas Tenbrake & Hannes Pfeifer

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Contributions

R.H.G. and L.-M.N. conceptualized experiments. B.S.S., Y.P. and A.J.B., along with L.T., performed the CO2 fibre ablation, with help from J.S., L.C.K., J.S.K. and H.P. L.-M.N., J.K.R. and S.W. constructed the cavities, with help from C.H.V. L.-M.N. and J.K.R. performed single-protein experiments and protein data analysis. D.S.-B. performed autocorrelation analysis. J.K.R. and L.-M.N. performed protein and DNA mixture experiments. C.S. and R.H.G. devised experiments to confirm the mechanism. C.S., J.K.R. and L.-M.N. performed LBW experiments, noise characterization and photothermal bandwidth quantification. C.S. and D.S.-B. performed simulations. A.J.F. and B.M. wrote the hardware code. Z.Z. and E.R.C. designed and Z.Z. purified DNA constructs. R.H.G., L.-M.N., C.S., J.K.R. and D.S.-B. wrote the manuscript.

Corresponding author

Correspondence to Randall H. Goldsmith.

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Competing interests

R.H.G. and L.-M.N. have submitted PCT/US2023/079347, ‘Methods and systems for detecting diffusing single particles’, filed 10 November 2023.

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Nature thanks Michel Orrit, Judith Su and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Fig. 1 Zoomed-in view of single-molecule signals.

Representative 44-ms traces of proteins streptavidin, carbonic anhydrase, aprotinin and Myc-tag perturbing the cavity mode in both transmission and reflection. Data collected in cavity one.

Extended Data Fig. 2 Linearity of peak count with concentration.

Number of detected peaks induced by Myc-tag as a function of the protein concentration. Error bars represent the standard deviation across datasets. This scaling demonstrates that the peaks are induced by interactions between biomolecules and the cavity mode. Data collected in cavity two.

Extended Data Fig. 3 Poisson distribution of single-molecule signal arrival times.

Statistical analysis of the time intervals between single-molecule events for proteins streptavidin, carbonic anhydrase and Myc-tag. This analysis shows mono-exponential decays illustrating the underlying Poisson behaviour of the experiment and providing evidence of single-molecule detection.

Extended Data Fig. 4 Detection of protein molecules in buffer.

Single-molecule transients of aprotinin are readily observed in PBS buffer, whereas the pure PBS buffer and pure water are devoid of single-molecule signals.

Extended Data Fig. 5 Detection of signals sampled at 500 kHz.

Single diffusion events of <1 nm protein, Myc-tag, are resolvable with 2 μs temporal resolution. Representative single Myc-tag events collected with 500 kHz acquisition frequency. Data collected using cavity two at a concentration of 1 pM.

Extended Data Fig. 6 Histograms of detection events in mixtures.

a–c, Histograms showing extracted temporal widths and peak prominences of transmitted signals of a binary DNA Y-junction:duplex mixture (a; Supplementary Table 1), the mixture spiked with extra amounts of the pure Y-junction (b) and the mixture spiked with extra amounts of pure duplex (c).

Extended Data Fig. 7 Staggered detection from mixtures.

a,b, Scatter plots of peak prominence versus peak number showing consistency of molecular populations throughout the duration of an experiment and minimal cavity mode wandering for a binary mixture of aprotinin and Myc-tag (a) and a binary mixture of a DNA Y-junction and duplex (b; Supplementary Table 1).

Extended Data Fig. 8 Simulation of microcavity resonant mode.

Simulated cavity mode given the mirror properties of cavity one (Supplementary Table 5) showing the normalized square of the electric field, which is proportional to the total energy stored in the cavity. The zoomed image shows a particle localized in an antinode in the centre of the optical mode.

Extended Data Fig. 9 Photothermally distorted cavity resonance scan.

Photothermal-induced broadening of the transmitted cavity resonance in water, collected under cavity-length tuning at two different pump wavelengths. The blueshift in pump wavelength shifts the resonance position to lower piezo ramp voltage, demonstrating that increased piezo ramp voltage corresponds to increased cavity length. Furthermore, the direction of the broadening is indicative of a negative thermo-optic coefficient of the medium, as is expected in water. Despite the low circulating power (5.5 mW), photothermal broadening was apparent and enabled the high sensitivity of this measurement. The smaller peaks originate from polarization splitting owing to the birefringence of the cavity mode. Data collected with cavity four.

Extended Data Fig. 10 Replication of single-molecule-like signals by pulsing the length of the microcavity.

a, Transmitted intensity of locked cavity showing perturbations to the lock when voltage pulses are applied to the piezos at a frequency of 1 Hz. b, The applied pulse, input power and cavity-locking parameters can be optimized to mimic signals induced by diffusing molecules. a–d, The step-up voltage (grey) produced a steep reduction of the locked transmission signal as a result of the photothermal effect (a). This was followed by a brief recovery to the locked state by the PI feedback loop (b), followed by a second descent of the transmission signal as the step-up voltage of the pulse shifts the cavity in the opposite direction (c). Finally, the PI control recovers the locked state (d). Data collected with cavity four.

Supplementary information

Supplementary Information

This file contains DNA samples, simulations and calculations, additional discussion, Supplementary Tables 1–5, Supplementary Figures 1–20 and supplementary references.

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Needham, LM., Saavedra, C., Rasch, J.K. et al. Label-free detection and profiling of individual solution-phase molecules. Nature (2024). https://doi.org/10.1038/s41586-024-07370-8

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