Ultraviolet optical horn antennas for label-free detection of single proteins

Abstract

Single-molecule fluorescence techniques have revolutionized our ability to study proteins. However, the presence of a fluorescent label can alter the protein structure and/or modify its reaction with other species. 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. Unfortunately, the low autofluorescence brightness of proteins has greatly challenged single molecule detection so far. Here we introduce optical horn antennas, a dedicated nanophotonic platform enabling the label-free detection of single proteins in the UV. This design combines fluorescence plasmonic enhancement, efficient collection up to 85° angle and background screening. We detect the UV autofluorescence from immobilized and diffusing single proteins, and monitor protein unfolding and dissociation upon denaturation. Optical horn antennas open up a unique and promising form of fluorescence spectroscopy to investigate single proteins in their native states in real time.

Ultraviolet Nanophotonics Enables Autofluorescence Correlation Spectroscopy on Label-Free Proteins with a Single Tryptophan

“…This is achieved because of the combination of (i) nanophotonic UV antenna to enhance the signal, (ii) detailed analysis to reduce the background intensity, and (iii) chemical photostabilizing agents to avoid fluorescence saturation. Our results provide guidelines on how to extend plasmonics into the UV regime − and further develop label-free single molecule spectroscopy. ,,− Earlier works using UV aluminum nanophotonics were restricted to proteins containing a large number of Trp residues such as β-galactosidase (156 Trps) , and streptavidin (24 Trps) . Here we improve the sensitivity by more than 1 order of magnitude, down to the single tryptophan level.…”

“…The 9× enhancement stands also in good agreement with our calibration using the UV fluorescent dye pterphenyl. 35 If we compare between the best results found for the horn antenna (single Trp brightness 70 cts/s, background 600 cts/s) and the confocal setup (single Trp 8 cts/s, background 2000 cts/s), our combined solution improves the signal to background ratio by 30×. The 1000× lower detection volume with the antenna efficiently eliminates the background intensity stemming from the solution (Figure S6) yet at the expense of a supplementary background from the antenna luminescence (Figure 1i).…”

“…We use a 266 nm deep UV laser excitation and a fluorescence collection in the 310–360 nm range for our time-resolved UV confocal microscope (Figure b). To maximize the autofluorescence signal, we employ an optical horn antenna developed recently in our group (Figure c) . This nanophotonic device combines a central metal nanoaperture together with a conical horn microreflector.…”

Plasmonics for advance single-molecule fluorescence spectroscopy and imaging in biology

“…In addition, since field intensity relative to the aperture decay as a function of the axial distance, the axial positions of fluorophores was also determined in this work by utilizing the brightness-to-axialdistance relationship provided by antennas. Finally, in 2022, Barulin and colleagues introduce a new an optical horn antenna platform for single label-free proteins detection in the UV range via their natural ultraviolet fluorescence (Barulin et al, 2022). This antenna combines a conical horn reflector for fluorescence collection at ultrahigh angles with a metal nanoaperture for fluorescence enhancement and background screening.…”

Wireless at the Nanoscale: Towards Magnetically Tunable Beam Steering

“…To develop our idea, we use a well-known nanostructure, namely horn-like aperture plasmonic nanoantenna, which has been used for optical wireless communications 22 and plasmonic biosensing applications. 23 The system can be considered built by a noble metal (or ferromagnetic metal) film of thickness t, with a horn-like nanoaperture, placed on a conventional SiO 2 glass substrate, as illustrated in Fig. 2.…”