Signal Enhancement Enables Study of Label-Free Proteins

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 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).