Pushing the Frontiers of Single-Molecule Fluorescence Detection with Nanophotonics

Brief summary:
Our aim is to advance single-molecule detection beyond the traditional limits imposed by optical diffraction. By integrating nanophotonic components into advanced microscopes, we enhance the fluorescence brightness of single molecules and extend their domain of detection. Our ultimate goal is to watch molecules better, faster and with higher spatio-temporal resolutions.

Background motivation:
Because of the size mismatch between the wavelength of light (~500 nm) and the dimensions of a single molecule (~a few nm), the light-molecule interaction remains very inefficient and diffraction-limited. Two major limitations emerge: (1) the fluorescence brightness (detected photons per second and per molecule) remains low, challenging the detection and limiting the temporal resolution; and (2) single-molecule detection requires a narrow concentration window in the nanomolar range, high enough for a detectable signal, yet low enough to maintain molecular counting abilities. Our goal is to break these limits to further advance single-molecule detection.

Methods:
We use nanophotonic structures to confine light in nanoscale dimensions closer to the size of a single molecule. This nano-optical confinement enhances the fluorescence emission from individual molecules and enables new levels of control over its spatial, spectral, and temporal characteristics. We combine these structures with cutting-edge time-resolved fluorescence spectroscopy techniques to probe single molecules with unprecedented sensitivity and detail.

- Photonic nanostructures involved: zero-mode waveguide nanoapertures, horn antennas, plasmonic nanogap antennas, plasmonic nanoparticles, photonic nanojet

- Spectroscopy techniques used: fluorescence correlation spectroscopy and related techniques FCS, FCCS and FLCS, single molecule Förster resonant energy transfer smFRET, optical tweezers, confocal fluorescence microscopy

Impact:
At the interfaces between nanophotonics and biophotonics, our research advances the fundamental understanding of nanoscale light-matter interaction. It also contributes to the development of innovative techniques for the detection and analysis of biomolecules, with implications across molecular biology, biophysics, and analytical chemistry.

Highlights:

• Nanoantenna enhanced fluorescence: We developed plasmonic nanogap antennas that achieve world-leading performance in single-molecule fluorescence enhancement (Punj Nat. Nanotechnol. 2013; Flauraud Nano Lett. 2017) and revealed the presence of nanoscale heterogeneities in the outer membranes of living cells (Regmi Nano Lett. 2017).
• Zero-mode waveguide nanoapertures: We pioneered fluorescence enhancement using single metal nanoapertures, achieving over 1000-fold reduction in detection volume and significantly brighter emission. These nanophotonic devices were used to monitor DNA–peptide interactions at micromolar concentrations (Patra Nucleic Acids Res. 2021) and record fast single-molecule dynamics with microsecond resolution (Tiwari Adv. Opt. Mater. 2023; Nüesch JACS 2022).
• Label-free single protein detection with UV nanophotonics: We have developed UV nanophotonics and plasmonics for label-free detection of proteins (Barulin Nano Letters 2019, Roy ACS Nano 2023), and demonstrated the first ultraviolet detection of individual proteins (Barulin Nature Comm 2022) up to the ultimate limit of a single tryptophan residue (Roy Nano Letters 2023).
• Plasmonic nano-optical trapping: Our work has revealed the critical role of thermal effects in plasmonic nano-optical tweezers (Jiang Nano Lett. 2020), offering new mechanisms for nanoscale manipulation. We applied these concepts in a novel resonant nanoantenna design capable of trapping individual colloidal quantum dots as small as 11 nm in diameter (Jiang Nano Lett. 2021).

Funding