Purcell radiative rate enhancement of label-free proteins with ultraviolet aluminum plasmonics

Abstract

The vast majority of proteins are intrinsically fluorescent in the ultraviolet, thanks to the emission from their tryptophan and tyrosine amino-acid constituents. However, the protein autofluorescence quantum yields are generally very low due to the prevailing quenching mechanisms by other amino acids inside the protein. This motivates the interest to enhance the radiative emission rate of proteins using nanophotonic structures. Although there have been numerous reports of Purcell effect and local density of optical states control in the visible range using single dipole quantum emitters, the question remains open to apply these concepts in the UV on real proteins containing several tryptophan and tyrosine amino acids arranged in a highly complex manner. Here, we report the first complete characterization of the Purcell effect and radiative rate enhancement for the UV intrinsic fluorescence of label-free β-galactosidase and streptavidin proteins in plasmonic aluminum nanoapertures. We find an excellent agreement with a calibration performed using a high quantum yield UV fluorescent dye. Demonstrating and intensifying the Purcell effect is essential for the applications of UV plasmonics and the label-free detection of single proteins.

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

“…For a description of the aluminum nanoaperture influence on the fluorescence process, our group has recently published a detailed characterization using label-free proteins in the UV. 35 As shown by the data in Fig. 1, the presence of the horn reflector improves the collection efficiency, but the fluorescence excitation and emission enhancements occurring in the nanoaperture are not affected.…”

“…A separate calibration using diffusing molecules shows that the average brightness per protein is 3× lower for streptavidin as compared to βgalactosidase. 35 Although streptavidin has a 6.5× lower absolute number of tryptophan residues, their average quantum yield is estimated to be around 3.5% while it is only of 1.6% in β-galactosidase due to a higher quenching by nearby aminoacids. 18,35 The experiments on immobilized streptavidin proteins are performed using a 5 nM concentration to work in the same conditions as for Fig.…”

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.…”

“…Therefore, the brightness for streptavidin is not 24× higher than the brightness for TNase. The average quantum yield for a Trp residue in streptavidin was estimated to be 3.5 ± 1%, 18,36 while the quantum yield of Trp in TNase was reported to be 28 ± 2%. 2,41 With these values, the brightness for streptavidin is expected to be 24 × 3.5/28 = 3.0 ± 0.9 times higher than the TNase brightness.…”

“…As the horn antenna is a weakly resonant structure (as compared to a dimer nanogap antenna , ), its gain is essentially brought by the local excitation intensity increase and the improved collection efficiency . The quantum yield enhancement plays a minor role here, so similar net fluorescence enhancement is expected although proteins with different Trp quantum yields are used. The 9× enhancement stands also in good agreement with our calibration using the UV fluorescent dye p -terphenyl .…”