Boosting tryptophan fluorescence with optical nanoantennas to watch label-free protein dynamics with single molecule resolution at high concentration

Project Information

TryptoBoost

Grant agreement ID: 723241

DOI

10.3030/723241

Project closed

EC signature date 25 April 2017

Start date 1 October 2017

End date 30 September 2023

Funded under

• EXCELLENT SCIENCE - European Research Council (ERC)

Total cost

€ 1 947 208,13

EU contribution

€ 1 947 208,00

Coordinated by

CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE CNRS
France

Periodic Reporting for period 4 - TryptoBoost (Boosting tryptophan fluorescence with optical nanoantennas to watch label-free protein dynamics with single molecule resolution at high concentration)

Reporting period: 2022-04-01 to 2023-09-30

Summary of the context and overall objectives of the project

Proteins execute a broad range of functions that are central to life. Understanding these functions ultimately requires experiments at the single protein level to reveal dynamics and heterogeneities hidden in ensemble-averaged measurements. Currently, the preferred method to study single protein machineries is based on fluorescence techniques. However, fluorescence experiments suffer from major challenges: the need for external fluorescent labeling, weak signals and low non-physiological concentrations in the nanomolar range. These challenges severely limit the applicability and biological relevance of single molecule fluorescence on proteins.

The TryptoBoost project aims to overcome all the previous challenges, and efficiently monitor single label-free proteins using their intrinsic tryptophan fluorescence enhanced by optical nanoantennas in the ultraviolet. Using the natural amino acid fluorescence rules out all drawbacks due to external labeling, while the optical nanostructures enable single protein analysis at the physiologically relevant micromolar concentrations thanks to the localization and enhancement of light-matter interactions at the nanoscale.

More than 90% of all the 40,000 human proteins contain some aromatic amino acids which are fluorescent in the ultraviolet. The project breakthroughs exploring the UV autofluorescence of proteins without any external label will benefit a broad range of biophysical, chemical, and medical applications. For instance, it will improve the development of therapeutic drugs, increase the detection sensitivity and read-out speed in analytical biosensing on chip, and provide new nanostructures to enhance ultraviolet photocatalysis.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

The TryptoBoost project represents a pioneering leap in the realms of UV fluorescence enhancement and label-free protein detection. It has successfully opened new horizons by demonstrating the enhancement of single protein UV fluorescence using aluminum zero-mode waveguide nanoapertures. This achievement enables researchers to study individual proteins in their natural state without the interference of external fluorescent markers. Moreover, the project has significantly improved protein photostability in the UV range through innovative strategies, marking a major milestone in the field. The introduction of an optical horn antenna platform has revolutionized real-time monitoring of protein behavior, offering unparalleled sensitivity and resolution. Furthermore, the project's advancements in the UV horn antenna design allow for the detection of single tryptophan residues, expanding the range of proteins that can be studied in this manner. Additionally, the utilization of rhodium nanocubes-based UV resonant nanogap antennas has substantially increased UV autofluorescence brightness, facilitating high-concentration UV autofluorescence correlation spectroscopy. These collective achievements not only advance scientific understanding but also hold immense promise for applications in biotechnology, pharmaceuticals, and medical diagnostics. The interdisciplinary approach of the project fosters collaboration and innovation across scientific disciplines, further enriching related fields.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

The TryptoBoost project has achieved remarkable progress beyond the state of the art, advancing research in UV fluorescence enhancement, label-free protein detection, and related fields. These achievements hold promises for diverse practical applications.

- Single Protein UV Fluorescence Enhancement: We broke new ground by demonstrating single protein UV fluorescence enhancement using aluminum zero-mode waveguide nanoapertures, enabling observation of proteins without external fluorescent markers—a pioneering feat in UV plasmonics.

- Enhanced Protein Photostability: Our innovative approach, involving oxygen scavengers and reducing agents, significantly improved protein photostability in the UV range, marking the first quantitative assessment of photostability improvement strategies for label-free UV proteins.

- Optical Horn Antenna Platform: The novel optical horn antenna platform offers unprecedented sensitivity and resolution for label-free UV protein detection. It enables real-time monitoring of protein behavior, advancing biochemical assays with single protein resolution.

- Single Tryptophan Detection: Advancements in the UV horn antenna design push sensitivity to the single tryptophan level, allowing the study of proteins with only a few tryptophan residues.

- UV Resonant Nanogap Antennas: Rhodium nanocubes-based UV resonant nanogap antennas significantly amplify UV autofluorescence, enhancing brightness by up to 120-fold and enabling UV autofluorescence correlation spectroscopy at high concentrations.

In related fields:

- Fluorescence Spectroscopy and Nanophotonics: Aluminum nanoapertures advance single molecule Förster resonance energy transfer studies and DNA-peptide interactions in visible spectral ranges.

- Nano-Optical Tweezers: Research quantifying temperature increases in nano-optical trapping optimizes thermal force for plasmonic trapping, achieving remarkable capabilities for nano-objects.

- Nanophotonic Devices Fabrication: Strategies counter UV photocorrosion of aluminum, exploring alternative materials and designs in nanophotonic device fabrication.

This interdisciplinary project, bridging physics, chemistry, and nanosciences, fosters collaboration and innovation at their intersection. These achievements underscore the remarkable progress made in the TryptoBoost project, promising transformative impact across scientific disciplines and applications.

Optical horn antenna for single label-free protein detection in the ultraviolet