![]() Here, we introduce an optical horn antenna platform for label-free detection of single proteins in the UV with unprecedented resolutions and sensitivity. Alternative designs must be developed to offer a highly efficient platform, reaching the needs of high photon count rates, microsecond time resolution, background-free operation, and full compatibility with the UV detection of proteins. Intense fluorescence enhancement factors have been achieved with strongly absorbing dyes in the visible 22– 27, but most optical antennas designs remain unsuitable for UV protein detection due to their narrowband spectral response 28, 29, challenging nanofabrication 24, 27, or requirement for solid-state integration 30, 31. In analogy to radiofrequency antennas, optical antennas offer a way to control and intensify the emission of single quantum emitters 21. Collecting the forbidden UV light emitted at high angles is crucial to maximize the autofluorescence signal and unlock single label-free protein detection. UV objectives have a numerical aperture typically below 0.8, which corresponds to a maximum collection angle of 33° into the quartz substrate of 1.48 refractive index. In the UV, however, the choice of microscope objectives is strongly restricted 14. Microscopes operating in the visible spectral range use objectives of high numerical apertures of 1.4 or above to maximize the fluorescence collected from a single molecule. This fundamental phenomenon is known as supercritical or forbidden light 19, 20. One of the main limiting issues is that close to a planar dielectric interface, a significant fraction of the light from a single dipole is emitted at large angles above 65°. Hence, new nanotechnology tools need to be introduced to intensify the emission from single proteins. However, proteins are orders of magnitude dimmer as compared to conventional fluorescent dyes, so that single protein UV detection has remained a major challenge so far 16– 18. Being able to detect the UV autofluorescence from a single (label-free) protein would be a disruptive method offering many benefits of fluorescence techniques (signal-to-noise ratio, temporal dynamics, sensitivity…) without introducing the labeling drawbacks. More than 90% of all proteins contain some tryptophan or tyrosine aminoacid residues which are naturally fluorescent in the UV 15. ![]() The protein autofluorescence in the ultraviolet (UV) is an appealing route to rule out all the issues related to external fluorescence labeling 14, 15. Therefore, label-free alternatives to detect single molecules are actively investigated 9– 13. While single-molecule fluorescence techniques have achieved impressive results towards this goal 1, 2, the requirement for fluorescent markers can potentially lead to severe issues altering the protein structure or modifying its reaction with other species 3– 8. ![]() ![]() One of the ultimate goals of molecular biology is to watch how single proteins work in their native state. Optical horn antennas open up a unique and promising form of fluorescence spectroscopy to investigate single proteins in their native states in real time. We detect the UV autofluorescence from immobilized and diffusing single proteins, and monitor protein unfolding and dissociation upon denaturation. This design combines fluorescence plasmonic enhancement, efficient collection up to 85° angle and background screening. ![]() Here we introduce optical horn antennas, a dedicated nanophotonic platform enabling the label-free detection of single proteins in the UV. Unfortunately, the low autofluorescence brightness of proteins has greatly challenged single molecule detection so far. To avoid the need for a fluorescent label, the intrinsic autofluorescence of proteins in the ultraviolet offers the benefits of fluorescence techniques without introducing the labelling drawbacks. However, the presence of a fluorescent label can alter the protein structure and/or modify its reaction with other species. Single-molecule fluorescence techniques have revolutionized our ability to study proteins. ![]()
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