Carlos Saavedra

Label-free single-molecule detection schemes can provide key molecular information. Here, we deploy the elevated light-matter interaction in locked high-finesse fiber-based Fabry-Pérot microcavities (FFPCs) to push label-free detection to the single-amino acid limit, even to the smallest amino acid, glycine. Varying the sensitivity of the locked FFPC allows differentiation of subsets of amino acids at the single-molecule level, with examples including the differentiation of a small peptide from tryptophan, tryptophan from histidine, histidine from alanine, and phosphorylated threonine from threonine. Molecular detection events are visible as neuron-like signal bursts of decreased FFPC transmission, which can be replicated by simulations that include non-linear coupling between optical and thermal parameters of the locked FFPC. Importantly, differentiation was achieved in solution at pM concentrations, making the detection process suitable for integration with single-molecule processive-protein degradation. Thus, this combination of sensitivity and differentiating ability makes FFPCs a promising new photonic technology element for protein sequencing.

Fiber Fabry-Perot microcavities (FFPCs) enhance light-matter interactions by localizing light in time and space. Such FFPCs are at the heart of this powerful detection scheme exploiting photothermal nonlinearities and Pound-Drever-Hall frequency locking that enabled label-free profiling of single solution-phase biomolecules with unprecedented sensitivity. Here, we deploy a combination of experiment and simulation to provide a quantitative mechanism for the observed single-molecule sensitivity and achieve quantitative agreement with experiment. A key element of the mechanism is maintaining the FFPC in an unstable regime and allowing it to rapidly shift between hot and cold photothermal equilibria upon perturbation. We show how Brownian molecular trajectories, introducing resonance fluctuations less than 1000th of the already narrow microcavity line width, can produce selective and highly amplified responses. Such perturbations are found to exist in a specific and tunable frequency window termed the molecular velocity filter window. The model's predictive capacity suggests it will be an important tool to identify additional modes of sensitivity to single-molecule hydrodynamic behavior.

Fiber Fabry-Perot microcavities (FFPCs) enhance light-matter interactions by localizing light in time and space. A new detection scheme exploiting photothermal non-linearities and Pound-Drever-Hall frequency locking enabled label-free detection of solution-phase single biomolecules with unprecedented sensitivity. Here, we deploy a combination of experiment and simulation to provide a quantitative mechanism for the observed single-molecule sensitivity and achieve quantitative agreement with experiment. Key elements of the mechanism include maintaining the FFPC in an unstable regime, allowing it to rapidly shift to hot and cold pho-tothermal equilibria upon perturbation. We show how Brownian molecular trajectories introducing resonance fluctuations less than one thousandth of a linewidth can produce selective and highly amplified responses as long as the perturbations exist in a specific and tunable frequency window termed the molecular velocity filter window. The model’s predictive capacity suggest it will be an im-portant tool to identify new regimes of single-molecule hydrodynamic profiling.

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 method^{2,3,4,5,6,7,8,9,10,11,12,13,14,15,16} capable of revealing details of molecular conformation in solution^17,18 would allow a new microscopic perspective of unprecedented detail. Here we use the enhanced light–molecule interactions in high-finesse fibre-based Fabry–Pérot microcavities^19,20,21 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 dynamics^22,23 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.

Fiber-Fabry-Perot microcavities are tools that enhance light-matter interactions. A new detection scheme exploiting photo-thermal effects and Pound-Drever-Hall locking technique enabled label-free detection of solution-phase single biomolecules hundred times smaller than the wavelength in solution phase.

We present three high finesse tunable monolithic fiber Fabry-Perot cavities (FFPCs) with high passive mechanical stability. The fiber mirrors are fixed inside slotted glass ferrules, which guarantee an inherent alignment of the resonators. An attached piezoelectric element enables fast tuning of the FFPC resonance frequency over the entire free-spectral range for two of the designs. Stable locking of the cavity resonance is achieved for sub-Hertz feedback bandwidths, demonstrating the high passive stability. At the other limit, locking bandwidths up to tens of kilohertz, close to the first mechanical resonance, can be obtained. The root-mean-square frequency fluctuations are suppressed down to ∼2% of the cavity linewidth. Over a wide frequency range, the frequency noise is dominated by the thermal noise limit of the system’s mechanical resonances. The demonstrated small footprint devices can be used advantageously in a broad range of applications like cavity-based sensing techniques, optical filters or quantum light-matter interfaces.