"Protein Analysis Gets a Boost: Researchers Develop Advanced Label-Free Technique Using Ultraviolet Nanophotonics"

Scientists from Institut Fresnel, Aix Marseille University, have made a significant breakthrough in the field of protein study using ultraviolet nanophotonics to enable autofluorescence correlation spectroscopy on label-free proteins with a single Tryptophan.

Using the ultraviolet autofluorescence of tryptophan amino acids offers fascinating perspectives to study single proteins without the drawbacks of fluorescence labeling. However, the low autofluorescence signals have so far limited the UV detection to large proteins containing several tens of tryptophan residues. This limit is not compatible with the vast majority of proteins which contain only a few tryptophans.

The team has successfully pushed the sensitivity of label-free ultraviolet fluorescence correlation spectroscopy (UV-FCS) down to the single tryptophan level. The combination of nanophotonic plasmonic antennas, antioxidants, and background noise reduction techniques has improved the signal-to-background noise ratio by over an order of magnitude and enabled UV-FCS on TNase proteins with a single tryptophan residue.

This sensitivity breakthrough unlocks the applicability of the UV-FCS technique to a broad library of label-free proteins. This research is significant as it allows for proteins to be studied in their native state without introducing any external fluorescent label, thus simplifying the protein preparation and purification steps and ruling out the necessary controls to ensure the fluorescent label does not affect the protein.

This research opens up new opportunities for the study of proteins in the deep ultraviolet (UV) range using the natural fluorescence of tryptophan, tyrosine, and phenylalanine amino acids. The researchers have provided guidelines on how to extend plasmonics into the UV regime and further develop label-free single-molecule spectroscopy.

In a recent Nano Letters publication, our team breaks into this sensitivity limit and achieves label-free UV-autofluorescence detection down to the single tryptophan level thanks to a nanophotonic enhancement of the signal. Our approach relies on a rationally-designed combination of plasmonic antennas, antioxidants, and background noise reduction techniques to improve the signal-to-background ratio by over an order of magnitude. Achieving the ultimate sensitivity of UV-FCS down to the single tryptophan regime has wide applications for various communities from nanophotonics to biochemistry.

More information:
1.Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 3rd ed.; Springer US, 2006; pp 529– 575.

2.Arroyo, J. O.; Kukura, P. Non-Fluorescent Schemes for Single-Molecule Detection, Imaging and Spectroscopy. Nat. Photonics 2016, 10, 11– 17, DOI: 10.1038/nphoton.2015.251

3.Li, Q.; Seeger, S. Label-Free Detection of Protein Interactions Using Deep UV Fluorescence Lifetime Microscopy. Anal. Biochem. 2007, 367, 104– 110, DOI: 10.1016/j.ab.2007.04.050

4.Barulin, A.; Claude, J.-B.; Patra, S.; Bonod, N.; Wenger, J. Deep Ultraviolet Plasmonic Enhancement of Single Protein Autofluorescence in Zero-Mode Waveguides. Nano Lett. 2019, 19, 7434– 7442, DOI: 10.1021/acs.nanolett.9b03137

Provided by Aix-Marseille University