Preventing Corrosion of Aluminum Metal with Nanometer-Thick Films of Al2O3 Capped with TiO2 for Ultraviolet Plasmonics

Extending plasmonics into the ultraviolet range imposes the use of aluminum to achieve the best optical performance. However, water corrosion is a major limiting issue for UV aluminum plasmonics, as this phenomenon occurs significantly faster in presence of UV light, even at low laser powers of a few microwatts. Here we assess the performance of nanometer-thick layers of various metal oxides deposited by atomic layer deposition (ALD) and plasma-enhanced chemical vapor deposition (PECVD) on top of aluminum nanoapertures to protect the metal against UV photocorrosion. The combination of a 5 nm Al2O3 layer covered by a 5 nm TiO2 capping provides the best resistance performance, while a single 10 nm layer of SiO2 or HfO2 is a good alternative. We also report the influence of the laser wavelength, the laser operation mode and the pH of the solution. Properly choosing these conditions significantly extends the range of optical powers for which the aluminum nanostructures can be used. As application, we demonstrate the label-free detection of streptavidin proteins with improved signal to noise ratio. Our approach is also beneficial to promote the long-term stability of the aluminum nanostructures. Finding the appropriate nanoscale protection against aluminum corrosion is the key to enable the development of UV plasmonic applications in chemistry and biology

La détection de fluorescence des protéines individuelles sans marquage dans l'UV améliorée par les nanostuctures plasmoniques d'aluminium

Les techniques de fluorescence de molécule individuelle permettent de suivre la dynamique moléculaire et les interactions dans les processus biologiques. Maintenant, la dynamique moléculaire des protéines est principalement accompagnée du marquage fluorescent externe. Cependant, une molécule attachée peut perturber la dynamique de protéines. Heureusement, la majorité des protéines contiennent le tryptophane ou la tyrosine qui absorbent et émettent la lumière dans le domaine spectral d'UV entre 260 nm et 400 nm. Ces acides aminés ont de basses efficacités quantiques, photostabilisées dans l'UV et la section efficace d'absorption, qui gênent la détection des protéines individuelles. Afin d'atteindre la sensitivité de l'auto fluorescence UV des protéines individuelles, nous développons un microscope confocal UV à la résolution temporelle avec les lasers de 266 nm et 295 nm. Nous quantifions la sensitivité de détection et l'effet des techniques de photostabilisation sur l'autofluorescence des protéines. La spectroscopie de corrélation de fluorescence (SCF) et les mesures de time-correlated single photon counting (TCSPC) fournissent des informations quantitatives du volume de détection, de l'amélioration de fluorescence (AF), et de la photokinétique accélérée des molécules émettant à la présence et à l'absence des nanostructures d'aluminium (Al). En utilisant le p-terphenyl, nous optimisons les nanostructures plasmoniques d'Al afin d'améliorer la fluorescence. Sous certaines conditions, le confinement de la lumière et l'AF dans les structures d'Al permettent d'appliquer la plasmonique UV pour la détection des protéines individuelles de beta-galactosidase sans marquageSingle molecule fluorescence techniques enable to monitor the molecular dynamics and interactions in the biological processes. Nowadays, the molecular dynamics of proteins is principally accompanied by external fluorescent labeling. However, an attached molecule might perturb the protein dynamics. Fortunately, a vast majority of proteins contain tryptophan and tyrosine that absorb and emit light in the UV range of 260-400 nm. These intrinsically fluorescent amino acids yield limited absorption cross-section, quantum yield, and photostability in the UV range, which hampers single protein UV autofluorescence detection. In order to reach single molecule sensitivity of protein UV autofluorescence, we develop a time-resolved UV confocal microscope with 266 nm and 295 nm excitations and the detection optics in the near UV. Based on the total fluorescence time traces, we quantify the single molecule sensitivity, the effect of photostabilization techniques on the protein autofluorescence. Fluorescence correlation spectroscopy (FCS) and time-correlated single photon counting (TCSPC) measurements provide quantitative information on the detection volume, the fluorescence enhancement factors, and the accelerated photokinetics of the UV emitting molecules in the presence and absence of the aluminum (Al) nanostructures. Using p-terphenyl as a bright UV emitting molecule, we optimize the Al plasmonic nanostructures to enhance the single molecule fluorescence. Under certain conditions, the light confinement and fluorescence enhancement in the aluminum nanostructures enable to apply the UV plasmonics for the single molecule detection of label-free beta-galactosidase protei

Ultraviolet Photostability Improvement for Autofluorescence Correlation Spectroscopy on Label-Free Proteins

International audienceThe poor photostability and low brightness of protein autofluorescence have been major limitations preventing the detection of label-free proteins at the single molecule level. Overcoming these issues, we report here a strategy to promote the photostability of proteins and use their natural tryptophan autofluorescence in the ultraviolet (UV) for fluorescence correlation spectroscopy (FCS). Combining enzymatic oxygen scavengers with antioxidants and triplet state quenchers greatly promotes the protein photostability, reduces the photobleaching probability and improves the net autofluorescence detection rate. Our results show that the underlying photochemical concepts initially derived for organic visible fluorescent dyes are quite general. Using this approach, we achieved UV fluorescence correlation spectroscopy on label-free streptavidin proteins containing only 24 tryptophan residues, 6.5× less than the current state-of-the-art. This strategy greatly extends the possibility to detect single label-free proteins with the versatility of single molecule fluorescence without requiring the presence of a potentially disturbing external fluorescent marker. It also opens new perspectives to improve the UV durability of organic devices

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

International audienceThe 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

International audienceSingle-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

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

International audienceThe 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

Deep Ultraviolet Plasmonic Enhancement of Single Protein Autofluorescence in Zero-Mode Waveguides

International audienceSingle molecule detection provides detailed information about molecular structures and functions, but it generally requires the presence of a fluorescent marker which can interfere with the activity of the target molecule or complicate the sample production. Detecting a single protein with its natural UV autofluorescence is an attractive approach to avoid all the issues related to fluorescence labelling. However, the UV autofluorescence signal from a single protein is generally extremely weak. Here, we use aluminum plasmonics to enhance the tryptophan autofluorescence emission of single proteins in the UV range. Zero-mode waveguides nanoapertures enable observing the UV fluorescence of single label-free β-galactosidase proteins with increased brightness, microsecond transit times and operation at micromolar concentrations. We demonstrate quantitative measurements of the local concentration, diffusion coefficient and hydrodynamic radius of the label-free protein over a broad range of zero-mode waveguide diameters. While the plasmonic fluorescence enhancement has generated a tremendous interest in the visible and near-infrared parts of the spectrum, this work pushes further the limits of plasmonic-enhanced single molecule detection into the UV range and constitutes a major step forward in our ability to interrogate single proteins in their native state at physiological concentrations

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

International audienceUsing the ultraviolet autofluorescence of tryptophan aminoacids offers fascinating perspectives to study single proteins without the drawbacks of fluorescence labelling. 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. Here we push the sensitivity of label-free ultraviolet fluorescence correlation spectroscopy (UV-FCS) down to the single tryptophan level. Our results show how the combination of nanophotonic plasmonic antennas, antioxidants and background reduction techniques can improve the signal-to-background ratio by over an order of magnitude and enable UV-FCS on thermonuclease proteins with a single tryptophan residue. This sensitivity breakthrough unlocks the applicability of UV-FCS technique to a broad library of label-free proteins

Zero-mode waveguides can be made better: fluorescence enhancement with rectangular aluminum nanoapertures from the visible to the deep ultraviolet

International audienceNanoapertures milled in metallic films called zero-mode waveguides (ZMWs) overcome the limitations of classical confocal microscopes by enabling single molecule analysis at micromolar concentrations with improved fluorescence brightness. While the ZMWs have found many applications in single molecule fluorescence studies, their shape has been mainly limited to be circular. Owing to the large parameter space to explore and the lack of guidelines, earlier attempts using more elaborate shapes have led to unclear conclusions whether or not the performance was improved as compared to a circular ZMW. Here, we comparatively analyze the performance of rectangular-shaped nanoapertures milled in aluminum to enhance the fluorescence emission rate of single molecules from the near infrared to the deep ultraviolet. Our new design is based on rational principles taking maximum advantage of the laser linear polarization. While the long edge of the nanorectangle is set to meet the cutoff size for the propagation of light into the nanoaperture, the short edge is reduced to 30 nm to accelerate the photodynamics while maintaining bright fluorescence rates. Our results show that both in the red and in the ultraviolet, the nanorectangles provide 50% brighter photon count rates as compared to the best performing circular ZMWs and achieve fluorescence lifetimes shorter than 300 ps. These findings can be readily used to improve the performance of ZMWs, especially for fast biomolecular dynamics, bright single-photon sources, and ultraviolet plasmonics