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Signal Enhancement Enables Study of Label-Free Proteins

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Researchers at Institut Fresnel have developed a technique to detect the ultraviolet-autofluorescence signal in single proteins, opening the way for the label-free study of thousands of proteins whose natural fluorescence cannot be detected using existing technology.

Though proteins are fluorescent in the UV because they contain tryptophan amino acids, most proteins have only one to five tryptophan — which is too few to provide a strong UV signal at the level required for label-free protein detection. The weak fluorescence signals and large backgrounds found in most proteins have limited label-free UV detection techniques to the few large proteins that contain several tens of tryptophan residues.

Using a combination of plasmonic antennas, antioxidants, and background reduction techniques, the Institut Fresnel researchers, led by Jérôme Wenger, improved the signal-to-background ratio (SBR) in UV-autofluorescence proteins by more than one order of magnitude — enough to enable label-free detection. The researchers used both nanophotonic and plasmonic elements to enhance the fluorescence, and antioxidants to neutralize the reactive oxygen species ubiquitous to UV. The approach used by the team for the suppression of background noise was based on the researchers’ rational understanding of the physical origins of the background.

The researchers used this framework to enhance the sensitivity of UV-fluorescence correlation spectroscopy (UV-FCS), enabling it to be used to achieve label-free UV-autofluorescence detection down to the single tryptophan level. The team demonstrated UV-sensitive detection using UV-FCS on thermonuclease proteins with a single tryptophan residue.
Researchers pushed the sensitivity of label-free ultraviolet fluorescence correlation spectroscopy (UV-FCS) down to the single tryptophan level. Courtesy of Prithu Roy, Jean-Benoît Claude, Sunny Tiwari, Aleksandr Barulin, and Jérôme Wenger, ’Ultraviolet Nanophotonics Enables Autofluorescence Correlation Spectroscopy on Label-Free Proteins with a Single Tryptophan” Nano Letters 2023.
Researchers in France have pushed the sensitivity of label-free ultraviolet fluorescence correlation spectroscopy (UV-FCS) down to the single tryptophan level. Courtesy of Prithu Roy et al., 'Ultraviolet Nanophotonics Enables Autofluorescence Correlation Spectroscopy on Label-Free Proteins with a Single Tryptophan,' Nano Letters, 2023.
The researchers’ said that the technique can be applied to a broad library of proteins that were not accessible previously through label-free methods. Over 90% of human proteins have at least one tryptophan residue, but only 4% have more than 20 tryptophan. UV-autofluorescence of tryptophan amino acids in proteins will allow scientists to study single proteins without incurring the drawbacks of fluorescence labeling.


Several scientific communities, from nanophotonics to biochemistry, can benefit from the technique to extend the sensitivity of UV-FCS down to the single tryptophan regime. Fluorescence correlation spectroscopy and related techniques could have a powerful impact on molecular biophysics, especially in the evaluation of diffusion properties, local concentrations, and kinetic reaction rates, according to the researchers.

Further, the signal-to-background maximization approach could be useful for scientists and engineers working with single-molecule fluorescence, photonics, or plasmonics.

The research was published in Nano Letters (www.doi.org/10.1021/acs.nanolett.2c03797).

Published: January 2023
Glossary
nano
An SI prefix meaning one billionth (10-9). Nano can also be used to indicate the study of atoms, molecules and other structures and particles on the nanometer scale. Nano-optics (also referred to as nanophotonics), for example, is the study of how light and light-matter interactions behave on the nanometer scale. See nanophotonics.
nanophotonics
Nanophotonics is a branch of science and technology that explores the behavior of light on the nanometer scale, typically at dimensions smaller than the wavelength of light. It involves the study and manipulation of light using nanoscale structures and materials, often at dimensions comparable to or smaller than the wavelength of the light being manipulated. Aspects and applications of nanophotonics include: Nanoscale optical components: Nanophotonics involves the design and fabrication of...
fluorescence
Fluorescence is a type of luminescence, which is the emission of light by a substance that has absorbed light or other electromagnetic radiation. Specifically, fluorescence involves the absorption of light at one wavelength and the subsequent re-emission of light at a longer wavelength. The emitted light occurs almost instantaneously and ceases when the excitation light source is removed. Key characteristics of fluorescence include: Excitation and emission wavelengths: Fluorescent materials...
plasmonics
Plasmonics is a field of science and technology that focuses on the interaction between electromagnetic radiation and free electrons in a metal or semiconductor at the nanoscale. Specifically, plasmonics deals with the collective oscillations of these free electrons, known as surface plasmons, which can confine and manipulate light on the nanometer scale. Surface plasmons are formed when incident photons couple with the conduction electrons at the interface between a metal or semiconductor...
fluorescent protein
Fluorescent proteins are proteins that exhibit the property of fluorescence, which is the ability to absorb light at a specific wavelength and emit light at a longer wavelength. These proteins are widely used as molecular tags or markers in molecular and cellular biology to visualize and study the location, movement, and interactions of specific proteins within living cells or organisms. Key features and points about fluorescent proteins include: Fluorescence emission: Fluorescent proteins...
fluorescence correlation spectroscopy
A powerful method, referred to as FCS, for determining the average diffusion coefficients of fluorescent molecules in solution or membranes. FCS measurements rely on recording the transition of several thousands of molecules through the focal volume. The combination of short measurement times along with free positioning or scanning of the observation spot makes FCS an excellent tool for investigating diffusion heterogeneity over time and space.
autofluorescence
Autofluorescence refers to the natural emission of fluorescence exhibited by certain biological structures or molecules when exposed to light. Unlike fluorescence that results from the application of external fluorophores or dyes, autofluorescence arises intrinsically from endogenous molecules present in tissues or cells. Key points about autofluorescence: Endogenous emission: Autofluorescence occurs due to the presence of naturally fluorescent molecules within biological samples, such as...
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