Single-molecule Förster resonance energy transfer (smFRET) is widely used to monitor conformations and interaction dynamics at the molecular level. However, conventional smFRET measurements are ...ineffective at donor-acceptor distances exceeding 10 nm, impeding the studies on biomolecules of larger size. Here, we show that zero-mode waveguide (ZMW) apertures can be used to overcome the 10 nm barrier in smFRET. Using an optimized ZMW structure, we demonstrate smFRET between standard commercial fluorophores up to 13.6 nm distance with a significantly improved FRET efficiency. To further break into the classical FRET range limit, ZMWs are combined with molecular constructs featuring multiple acceptor dyes to achieve high FRET efficiencies together with high fluorescence count rates. As we discuss general guidelines for quantitative smFRET measurements inside ZMWs, the technique can be readily applied for monitoring conformations and interactions on large molecular complexes with enhanced brightness.
Aluminum can sustain plasmonic resonances down into the ultraviolet (UV) range to promote surface-enhanced spectroscopy and catalysis. Despite its natural alumina passivating layer, we find here that ...under 266 nm pulsed UV illumination, aluminum can undergo a dramatic photocorrosion in water within a few tens of seconds and even at low average UV powers. This aluminum instability in water environments is a critical limitation. We show that the aluminum photocorrosion is related to the nonlinear absorption by water in the UV range leading to the production of hydroxyl radicals. Different corrosion protection approaches are tested using scavengers for reactive oxygen species and polymer layers deposited on top of the aluminum structures. Using optimized protection, we achieve a 10-fold increase in the available UV power range leading to no visible photocorrosion effects. This technique is crucial to achieve stable use of aluminum nanostructures enabling UV plasmonics in aqueous solutions.
Plasmonic optical nanoantennas offer compelling solutions for enhancing light-matter interactions at the nanoscale. However, until now, their focus has been mainly limited to the visible and ...near-infrared regions, overlooking the immense potential of the ultraviolet (UV) range, where molecules exhibit their strongest absorption. Here, we present the realization of UV resonant nanogap antennas constructed from paired rhodium nanocubes. Rhodium emerges as a robust alternative to aluminum, offering enhanced stability in wet environments and ensuring reliable performance in the UV range. Our results showcase the nanoantenna's ability to enhance the UV autofluorescence of label-free streptavidin and hemoglobin proteins. We achieve significant enhancements of the autofluorescence brightness per protein by up to 120-fold and reach zeptoliter detection volumes, enabling UV autofluorescence correlation spectroscopy (UV-FCS) at high concentrations of several tens of micromolar. We investigate the modulation of fluorescence photokinetic rates and report excellent agreement between the experimental results and numerical simulations. This work expands the applicability of plasmonic nanoantennas to the deep UV range, unlocking the investigation of label-free proteins at physiological concentrations.
Dielectric Mie resonators have attracted a great deal of attention over the past few years thanks to their remarkable capabilities in manipulating light propagation at the nanoscale. However, the ...practical implementation of technological products is still elusive. One of the important limits is the absence of a high-performing material and a fabrication method that can be easily integrated into modern microelectronic devices at affordable costs. Here, we provide theoretical and experimental evidence of an alternative semiconductor material, SiGe alloys, for dielectric Mie resonator applications. As a material compatible with the processing requirements of the semiconductor industry, it possesses comparable optical properties to its conventional Si-based counterpart at visible frequencies in spite of its higher optical losses. These dielectric resonant particles can be obtained over very large surfaces on arbitrary silica substrates via spontaneous solid state dewetting of ultrathin (<100 nm) SiGe amorphous layers. Furthermore, the polycrystalline SiGe islands can be precisely organized in uniform arrays featuring low size dispersion. As an example, we demonstrate SiGe-based Mie resonator arrays functioning as color pass-band filters across the full visible spectral range. The filters function both in transmission and diffusion and are fabricated using a methodology compatible with C-MOS implemetation.
Abstract
G-quadruplexes (GQs), a non-canonical form of DNA, are receiving a huge interest as target sites for potential applications in antiviral and anticancer drug treatments. The biological ...functions of GQs can be controlled by specifically binding proteins known as GQs binding proteins. Some of the GQs binding proteins contain an arginine and glycine-rich sequence known as RGG peptide. Despite the important role of RGG, the GQs-RGG interaction remains poorly understood. By single molecule measurements, the interaction dynamics can be determined in principle. However, the RGG–GQs interaction occurs at micromolar concentrations, making conventional single-molecule experiments impossible with a diffraction-limited confocal microscope. Here, we use a 120 nm zero-mode waveguide (ZMW) nanoaperture to overcome the diffraction limit. The combination of dual-color fluorescence cross-correlation spectroscopy (FCCS) with FRET is used to unveil the interaction dynamics and measure the association and dissociation rates. Our data show that the RGG–GQs interaction is predominantly driven by electrostatics but that a specific affinity between the RGG sequence and the GQs structure is preserved. The single molecule approach at micromolar concentration is the key to improve our understanding of GQs function and develop its therapeutic applications by screening a large library of GQs-targeting peptides and proteins.
Graphical Abstract
Graphical Abstract
Nanoaperture to study fast dynamics between G-quadruplexes and peptides.
Single-molecule Förster resonance energy transfer (FRET) is a versatile technique for probing the structure and dynamics of biomolecules even in heterogeneous ensembles. However, because of the ...limited fluorescence brightness per molecule and the relatively long fluorescence lifetimes, probing ultrafast structural dynamics in the nanosecond time scale has thus far been very challenging. Here, we demonstrate that nanophotonic fluorescence enhancement in zero-mode waveguides enables measurements of previously inaccessible low-nanosecond dynamics by dramatically improving time resolution and reduces data acquisition times by more than an order of magnitude. As a prototypical example, we use this approach to probe the dynamics of a short intrinsically disordered peptide that were previously inaccessible with single-molecule FRET measurements. We show that we are now able to detect the low-nanosecond correlations in this peptide, and we obtain a detailed interpretation of the underlying distance distributions and dynamics in conjunction with all-atom molecular dynamics simulations, which agree remarkably well with the experiments. We expect this combined approach to be widely applicable to the investigation of very rapid biomolecular dynamics.
Zero-mode waveguide (ZMW) nanoapertures are widely used to monitor single molecules beyond the range accessible to normal microscopes. However, several aspects of the ZMW influence on the ...photophysics of fluorophores remain inadequately documented and sometimes controversial. Here, we thoroughly investigate the ZMW influence on the fluorescence of single immobilized Cy3B and Alexa 647 molecules, detailing the interplays between brightness, lifetime, photobleaching time, the total number of emitted photons, and Förster resonance energy transfer (FRET). Despite the plasmonic-enhanced excitation intensity in the ZMW, we find that the photostability is preserved with similar photobleaching times as on the glass reference. Both the fluorescence brightness and the total number of photons detected before photobleaching are increased, with an impressive gain of nearly five times that found for Alexa 647 dyes. Finally, the single-molecule data importantly allow a loophole-free characterization of the ZMW influence on the FRET process. We show that the FRET rate constant is enhanced by 50%, demonstrating that nanophotonics can mediate the energy transfer. These results deepen our understanding of the fluorescence enhancement in ZMWs and are of immediate relevance for single-molecule biophysical applications.
In this work, mechanically, chemically, and thermally resistant broadband and broad-angle antireflection coatings were prepared on 10 cm diameter glass substrates combining sol–gel deposition with ...nanoimprint lithography. The coatings are composed of water-repellent methylated silica (Si4O7Me2) and exhibit a transverse refractive index gradient created by tapered, nipple-dimple, subwavelength nanostructures, featuring a record vertical aspect ratio of ∼1.7. The structure is composed of hexagonal arrays of nanopillars (∼200 nm height, ∼120 nm width) and holes (∼50 nm depth, ∼100 nm width) with a 270 nm pitch. The corresponding effective refractive index is between 1.2 and 1.26, depending on the fabrication conditions. Total transmission for double-face nanoimprint wafers reaches 96–97% in the visible range; it is limited by specular reflection and mostly by the intrinsic diffusion of the glass substrate. The antireflective effect is effective up to an ∼60° incidence angle. We address the robustness of the inorganic-based coating in various realistic and extreme conditions, comparing them to the organic perfluoropolyether (PFPE) counterpart (master reference). The sol–gel system is extremely stable at high temperature (up to 600 °C, against 200 °C for the polymer reference). Both systems showed excellent chemical stability, except in strong alkaline conditions. The inorganic nanostructure showed an abrasion resistance of more than 2 orders of magnitude superior to the polymer one with less than 20% loss of antireflective performance after 2000 rubbing cycles under an ∼2 N cm–2 pressure. This difference springs from the large elastic modulus of the sol–gel material combined with an excellent adhesion to the substrate and to the specific nipple-dimple conformation. The presence of holes allows maintaining a refractive index gradient profile even after tearing out part of the nanopillar population. Our results are relevant to applications where transparent windows with broadband and broad-angle transmission are needed, such as protective glasses on photovoltaic cells or C-MOS cameras.
Flexible and stretchable photonics are emerging fields aiming to develop novel applications where the devices need to conform to uneven surfaces or whenever lightness and reduced thickness are major ...requirements. However, owing to the relatively small refractive index of transparent soft matter including most polymers, these materials are not well adapted for light management at visible and near-infrared frequencies. Here we demonstrate simple, low cost and efficient protocols for fabricating Si1−xGex-based, sub-micrometric dielectric antennas over record scales (50 mm wafers) with ensuing hybrid integration into different plastic supports. The transfer process has a near-unity yield: up to 99.94% for disordered structures and 99.5% for the ordered counterpart. Finally, we benchmark the optical quality of the dielectric antennas with light scattering measurements, demonstrating the control of the islands structural color and the onset of sharp Mie modes after encapsulation in plastic. Thanks to the ease of implementation of our fabrication methods, these results are relevant for the integration of SiGe-based dielectric Mie resonators in flexible substrates over large surfaces.
Abstract Background Recent studies suggest that patients with sickle cell disease (SCD) have profound vitamin D (VD) deficiency. Limited data exist on the effect of VD deficiency on bone fragility in ...these patients. Objectives To assess the prevalence of VD deficiency in adults with SCD and its consequences on bone metabolism and fragility. Methods This prospective study included 56 SCD adult patients (mean age 29.8 ± 9.5 years), in a clinically steady state. Clinical and laboratory data were recorded. Bone mineral density (BMD) was measured using dual X-ray absorptiometry. Fracture history, BMD, avascular osteonecrosis, H-shaped vertebra and markers of mineral metabolism were compared between two groups of patients presenting very low (≤ 6 ng/mL, n = 26) (group 1) and low (> 6 ng/mL, n = 26) (group 2) 25(OH)D concentration, respectively. Results Median 25(OH)D concentration was 6 ng/mL. VD deficiency (25(OH)D < 10 ng/mL) was found in 42 out of 56 patients (75%) and secondary hyperparathyroidism in 40 (71.4%). History of fracture was documented in 17 patients (30.3%), osteopenia and/or osteoporosis in 39.6% of patients. Overall, patients of group 1 were more likely to have sustained a fracture (42.8%) compared to patients of group 2 (17.8%) ( p = 0.04). These patients had also lower body mass index and significantly higher parathyroid hormone, C-terminal telopeptides of type I-collagen and bone-specific alkaline phosphatase serum levels. There was no difference between group for BMD, avascular osteonecrosis history, H-shaped vertebra, and disease severity markers. Conclusion This study suggests that VD deficiency is a key feature in SCD-bone disease. It is highly prevalent and associated with hyperparathyroidism, bone resorption markers, and history of fracture. The optimal supplementation regimen remains to be determined.
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