Fluorescence Enhancement in Topologically Optimized Gallium Phosphide All-Dielectric Nanoantennas.

Nanoantennas capable of large fluorescence enhancement with minimal absorption are crucial for future optical technologies from single-photon sources to biosensing. Efficient dielectric nanoantennas have been designed, however, evaluating their performance at the individual emitter level is challenging due to the complexity of combining high-resolution nanofabrication, spectroscopy and nanoscale positioning of the emitter. Here, we study the fluorescence enhancement in infinity-shaped gallium phosphide (GaP) nanoantennas based on a topologically optimized design. Using fluorescence correlation spectroscopy (FCS), we probe the nanoantennas enhancement factor and observe an average of 63-fold fluorescence brightness enhancement with a maximum of 93-fold for dye molecules in nanogaps between 20 and 50 nm. The experimentally determined fluorescence enhancement of the nanoantennas is confirmed by numerical simulations of the local density of optical states (LDOS). Furthermore, we show that beyond design optimization of dielectric nanoantennas, increased performances can be achieved via tailoring of nanoantenna fabrication.

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

Using the ultraviolet autofluorescence of tryptophan amino acids offers fascinating perspectives to study single proteins without the drawbacks of fluorescence labeling. 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.

Insights into animal septins using recombinant human septin octamers with distinct SEPT9 isoforms.

Septin GTP-binding proteins contribute essential biological functions that range from the establishment of cell polarity to animal tissue morphogenesis. Human septins in cells form hetero-octameric septin complexes containing the ubiquitously expressed SEPT9 subunit (also known as SEPTIN9). Despite the established role of SEPT9 in mammalian development and human pathophysiology, biochemical and biophysical studies have relied on monomeric SEPT9, thus not recapitulating its native assembly into hetero-octameric complexes. We established a protocol that enabled, for the first time, the isolation of recombinant human septin octamers containing distinct SEPT9 isoforms. A combination of biochemical and biophysical assays confirmed the octameric nature of the isolated complexes in solution. Reconstitution studies showed that octamers with either a long or a short SEPT9 isoform form filament assemblies, and can directly bind and cross-link actin filaments, raising the possibility that septin-decorated actin structures in cells reflect direct actin-septin interactions. Recombinant SEPT9-containing octamers will make it possible to design cell-free assays to dissect the complex interactions of septins with cell membranes and the actin and microtubule cytoskeleton.

Fast interaction dynamics of G-quadruplex and RGG-rich peptides unveiled in zero-mode waveguides.

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.

Single-molecule Detection of Ultrafast Biomolecular Dynamics with Nanophotonics.

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.

Single Photon Source from a Nanoantenna-Trapped Single Quantum Dot.

Single photon sources with high brightness and subnanosecond lifetimes are key components for quantum technologies. Optical nanoantennas can enhance the emission properties of single quantum emitters, but this approach requires accurate nanoscale positioning of the source at the plasmonic hotspot. Here, we use plasmonic nanoantennas to simultaneously trap single colloidal quantum dots and enhance their photoluminescence. The nano-optical trapping automatically locates the quantum emitter at the nanoantenna hotspot without further processing. Our dedicated nanoantenna design achieves a high trap stiffness of 0.6 (fN/nm)/mW for quantum dot trapping, together with a relatively low trapping power of 2 mW/µm2. The emission from the nanoantenna-trapped single quantum dot shows 7× increased brightness, 50× reduced blinking, 2× shortened lifetime, and a clear antibunching below 0.5 demonstrating true single photon emission. Combining nano-optical tweezers with plasmonic enhancement is a promising route for quantum technologies and spectroscopy of single nano-objects.

Quantifying the Role of the Surfactant and the Thermophoretic Force in Plasmonic Nano-optical Trapping.

Plasmonic nanotweezers use intense electric field gradients to generate optical forces able to trap nano-objects in liquids. However, part of the incident light is absorbed into the metal, and a supplementary thermophoretic force acting on the nano-object arises from the resulting temperature gradient. Plasmonic nanotweezers thus face the challenge of disentangling the intricate contributions of the optical and thermophoretic forces. Here, we show that commonly added surfactants can unexpectedly impact the trap performance by acting on the thermophilic or thermophobic response of the nano-object. Using different surfactants in double nanohole plasmonic trapping experiments, we measure and compare the contributions of the thermophoretic and the optical forces, evidencing a trap stiffness 20× higher using sodium dodecyl sulfate (SDS) as compared to Triton X-100. This work uncovers an important mechanism in plasmonic nanotweezers and provides guidelines to control and optimize the trap performance for different plasmonic designs.

Hyperuniform Monocrystalline Structures by Spinodal Solid-State Dewetting.

Materials featuring anomalous suppression of density fluctuations over large length scales are emerging systems known as disordered hyperuniform. The underlying hidden order renders them appealing for several applications, such as light management and topologically protected electronic states. These applications require scalable fabrication, which is hard to achieve with available top-down approaches. Theoretically, it is known that spinodal decomposition can lead to disordered hyperuniform architectures. Spontaneous formation of stable patterns could thus be a viable path for the bottom-up fabrication of these materials. Here, we show that monocrystalline semiconductor-based structures, in particular Si_{1-x}Ge_{x} layers deposited on silicon-on-insulator substrates, can undergo spinodal solid-state dewetting featuring correlated disorder with an effective hyperuniform character. Nano- to micrometric sized structures targeting specific morphologies and hyperuniform character can be obtained, proving the generality of the approach and paving the way for technological applications of disordered hyperuniform metamaterials. Phase-field simulations explain the underlying nonlinear dynamics and the physical origin of the emerging patterns.

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

Single 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 labeling. 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 waveguide nanoapertures enable the observation of the UV fluorescence of single label-free ß-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. Although 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.

Compressed perovskite aqueous mixtures near their phase transitions show very high permittivities: New prospects for high-field MRI dielectric shimming.

PURPOSE: Perovskites are greatly used nowadays in many technological applications because of their high permittivity, more specifically in the form of aqueous solutions, for MRI dielectric shimming. In this study, full dielectric characterizations of highly concentrated CaTiO3 /BaTiO3 water mixtures were carried out and new permittivity maxima was reached. METHODS: Permittivity measurements were done on aqueous solutions from 0%v/v to dry powder. The permittivity dependence with pressure was investigated. Scanning electron microscopy images were performed on a few representative solutions. BaTiO3 pressed pads of different thicknesses, permittivities, and distances to the head were compared in a 7T MRI scanner. RESULTS: Perovskite aqueous mixtures undergo a pressure-dependent phase transition in terms of permittivity, with increasing water content. A new relative permittivity maximum of 475 was achieved. Microscopic images revealed structural differences between phases. A B1+ improvement in the temporal lobe was obtained with thin, high permittivity BaTiO3 head. CONCLUSIONS: This new preparation method allows improved pad geometry and placement, as a result of the high relative permittivity values achieved. This method has great significance for medical applications of MRI dielectric shimming, being easy to replicate and implement on a large scale. Magn Reson Med 79:1753-1765, 2018. © 2017 The Authors Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine. This is an open access article under the terms of the Creative Commons Attribution NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.

Planar Optical Nanoantennas Resolve Cholesterol-Dependent Nanoscale Heterogeneities in the Plasma Membrane of Living Cells.

Optical nanoantennas can efficiently confine light into nanoscopic hotspots, enabling single-molecule detection sensitivity at biological relevant conditions. This innovative approach to breach the diffraction limit offers a versatile platform to investigate the dynamics of individual biomolecules in living cell membranes and their partitioning into cholesterol-dependent lipid nanodomains. Here, we present optical nanoantenna arrays with accessible surface hotspots to study the characteristic diffusion dynamics of phosphoethanolamine (PE) and sphingomyelin (SM) in the plasma membrane of living cells at the nanoscale. Fluorescence burst analysis and fluorescence correlation spectroscopy performed on nanoantennas of different gap sizes show that, unlike PE, SM is transiently trapped in cholesterol-enriched nanodomains of 10 nm diameter with short characteristic times around 100 µs. The removal of cholesterol led to the free diffusion of SM, consistent with the dispersion of nanodomains. Our results are consistent with the existence of highly transient and fluctuating nanoscale assemblies enriched by cholesterol and sphingolipids in living cell membranes, also known as lipid rafts. Quantitative data on sphingolipids partitioning into lipid rafts is crucial to understand the spatiotemporal heterogeneous organization of transient molecular complexes on the membrane of living cells at the nanoscale. The proposed technique is fully biocompatible and thus provides various opportunities for biophysics and live cell research to reveal details that remain hidden in confocal diffraction-limited measurements.

In-Plane Plasmonic Antenna Arrays with Surface Nanogaps for Giant Fluorescence Enhancement.

Optical nanoantennas have a great potential for enhancing light-matter interactions at the nanometer scale, yet fabrication accuracy and lack of scalability currently limit ultimate antenna performance and applications. In most designs, the region of maximum field localization and enhancement (i.e., hotspot) is not readily accessible to the sample because it is buried into the nanostructure. Moreover, current large-scale fabrication techniques lack reproducible geometrical control below 20 nm. Here, we describe a new nanofabrication technique that applies planarization, etch back, and template stripping to expose the excitation hotspot at the surface, providing a major improvement over conventional electron beam lithography methods. We present large flat surface arrays of in-plane nanoantennas, featuring gaps as small as 10 nm with sharp edges, excellent reproducibility and full surface accessibility of the hotspot confined region. The novel fabrication approach drastically improves the optical performance of plasmonic nanoantennas to yield giant fluorescence enhancement factors up to 104-105 times, together with nanoscale detection volumes in the 20 zL range. The method is fully scalable and adaptable to a wide range of antenna designs. We foresee broad applications by the use of these in-plane antenna geometries ranging from large-scale ultrasensitive sensor chips to microfluidics and live cell membrane investigations.

Single-step homogeneous immunoassay for detecting prostate-specific antigen using dual-color light scattering of metal nanoparticles.

Conventional sandwich-type immunoassays are widely used for protein biomarker detection, yet their workflows are challenged by the need for multiple incubation steps separated by washing cycles. Conducting these immunoassays is thus rather time-consuming and labor-intensive. Moreover, the limited sensitivity around 0.1 ng mL-1 is challenging the monitoring of cancer recurrence, for instance after radical prostatectomy in the case of prostate cancer. Here, we report a single-step homogeneous immunoassay using dual-color light scattering of metal nanoparticles. We detect human free prostate-specific antigen (f-PSA) in a buffered protein matrix solution with a single mixing and incubation step, no washing or purification cycle, and with a total experiment time of less than one hour. The limit of detection is 20 pg mL-1, which is 5× lower as compared to a conventional ELISA kit for f-PSA. The simpler, faster and more sensitive detection of cancer biomarkers opens promising opportunities to improve cancer diagnosis and health monitoring after cancer treatment.

Plasmonic Nanoantennas Enable Forbidden Förster Dipole-Dipole Energy Transfer and Enhance the FRET Efficiency.

Förster resonance energy transfer (FRET) plays a key role in biochemistry, organic photovoltaics, and lighting sources. FRET is commonly used as a nanoruler for the short (nanometer) distance between donor and acceptor dyes, yet FRET is equally sensitive to the mutual dipole orientation. The orientation dependence complicates the FRET analysis in biological samples and may even lead to the absence of FRET for perpendicularly oriented donor and acceptor dipoles. Here, we exploit the strongly inhomogeneous and localized fields in plasmonic nanoantennas to open new energy transfer routes, overcoming the limitations from the mutual dipole orientation to ultimately enhance the FRET efficiency. We demonstrate that the simultaneous presence of perpendicular near-field components in the nanoantenna sets favorable energy transfer routes that increase the FRET efficiency up to 50% for nearly perpendicular donor and acceptor dipoles. This new facet of plasmonic nanoantennas enables dipole-dipole energy transfer that would otherwise be forbidden in a homogeneous environment. As such, our approach further increases the applicability of single-molecule FRET over diffraction-limited approaches, with the additional benefits of higher sensitivities and higher concentration ranges toward physiological levels.

All-Dielectric Silicon Nanogap Antennas To Enhance the Fluorescence of Single Molecules.

Plasmonic antennas have a profound impact on nanophotonics as they provide efficient means to manipulate light and enhance light-matter interactions at the nanoscale. However, the large absorption losses found in metals can severely limit the plasmonic applications in the visible spectral range. Here, we demonstrate the effectiveness of an alternative approach using all-dielectric nanoantennas based on silicon dimers to enhance the fluorescence detection of single molecules. The silicon antenna design is optimized to confine the near-field intensity in the 20 nm nanogap and reach a 270-fold fluorescence enhancement in a nanoscale volume of λ(3)/1800 with dielectric materials only. Our conclusions are assessed by combining polarization resolved optical spectroscopy of individual antennas, scanning electron microscopy, numerical simulations, fluorescence lifetime measurements, fluorescence burst analysis, and fluorescence correlation spectroscopy. This work demonstrates that all-silicon nanoantennas are a valid alternative to plasmonic devices for enhanced single molecule fluorescence sensing, with the additional key advantages of reduced nonradiative quenching, negligible heat generation, cost-efficiency, and complementary metal-oxide-semiconductor (CMOS) compatibility.

Matching Nanoantenna Field Confinement to FRET Distances Enhances Förster Energy Transfer Rates.

Förster resonance energy transfer (FRET) is widely applied in chemistry, biology, and nanosciences to assess distances on sub-10 nm scale. Extending the range and applicability of FRET requires enhancement of the fluorescence energy transfer at a spatial scale comparable to the donor-acceptor distances. Plasmonic nanoantennas are ideal to concentrate optical fields at a nanoscale fully matching the FRET distance range. Here, we present a resonant aluminum nanogap antenna tailored to enhance single molecule FRET. A 20 nm gap confines light into a nanoscale volume, providing a field gradient on the scale of the donor-acceptor distance, a large 10-fold increase in the local density of optical states, and strong intensity enhancement. With our dedicated design, we obtain 20-fold enhancement on the fluorescence emission of donor and acceptor dyes, and most importantly up to 5-fold enhancement of the FRET rate for donor-acceptor separations of 10 nm. We also provide a thorough framework of the fluorescence photophysics occurring in the nanoscale gap volume. The presented enhancement of energy transfer flow at the nanoscale opens a yet unexplored facet of the various advantages of optical nanoantennas and provides a new strategy toward biological applications of single molecule FRET at micromolar concentrations.

FRET analysis of CP12 structural interplay by GAPDH and PRK.

CP12 is an intrinsically disordered protein playing a key role in the regulation of the Benson-Calvin cycle. Due to the high intrinsic flexibility of CP12, it is essential to consider its structural modulation induced upon binding to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK) enzymes. Here, we report for the first time detailed structural modulation about the wild-type CP12 and its site-specific N-terminal and C-terminal disulfide bridge mutants upon interaction with GAPDH and PRK by Förster resonance energy transfer (FRET). Our results indicate an increase in CP12 compactness when the complex is formed with GAPDH or PRK. In addition, the distributions in FRET histograms show the elasticity and conformational flexibility of CP12 in all supra molecular complexes. Contrarily to previous beliefs, our FRET results importantly reveal that both N-terminal and C-terminal site-specific CP12 mutants are able to form the monomeric (GAPDH-CP12-PRK) complex.

FRET enhancement in aluminum zero-mode waveguides.

Zero-mode waveguides (ZMWs) can confine light into attoliter volumes, which enables single molecule fluorescence experiments at physiological micromolar concentrations. Of the fluorescence spectroscopy techniques that can be enhanced by ZMWs, Förster resonance energy transfer (FRET) is one of the most widely used in life sciences. Combining zero-mode waveguides with FRET provides new opportunities to investigate biochemical structures or follow interaction dynamics at micromolar concentrations with single-molecule resolution. However, prior to any quantitative FRET analysis on biological samples, it is crucial to establish first the influence of the ZMW on the FRET process. Here, we quantify the FRET rates and efficiencies between individual donor-acceptor fluorophore pairs that diffuse into aluminum zero-mode waveguides. Aluminum ZMWs are important structures thanks to their commercial availability and the large amount of literature that describe their use for single-molecule fluorescence spectroscopy. We also compared the results between ZMWs milled in gold and aluminum, and found that although gold has a stronger influence on the decay rates, the lower losses of aluminum in the green spectral region provide larger fluorescence brightness enhancement factors. For both aluminum and gold ZMWs, we observed that the FRET rate scales linearly with the isolated donor decay rate and the local density of optical states. Detailed information about FRET in ZMWs unlocks their application as new devices for enhanced single-molecule FRET at physiological concentrations.

Nanophotonic enhancement of the Förster resonance energy-transfer rate with single nanoapertures.

Tailoring the light-matter interaction and the local density of optical states (LDOS) with nanophotonics provides accurate control over the luminescence properties of a single quantum emitter. This paradigm is also highly attractive to enhance the near-field Förster resonance energy transfer (FRET) between two fluorescent emitters. Despite the wide applications of FRET in nanosciences, using nanophotonics to enhance FRET has remained a debated and complex challenge. Here we demonstrate enhanced energy transfer within single donor-acceptor fluorophore pairs confined in single gold nanoapertures. Experiments monitoring both the donor and the acceptor emission photodynamics clearly establish a linear dependence of the FRET rate on the LDOS in nanoapertures, demonstrating that nanophotonics can be used to intensify the near-field energy transfer. Strikingly, we observe a significant six-fold increase in the FRET rate for large donor-acceptor separations exceeding 13 nm. Exciting opportunities are opened to investigate biochemical structures with donor-acceptor distances much beyond the classical Förster radius. Importantly, our approach is fully compatible with the detection of single biomolecules freely diffusing in water solution under physiological conditions.

Unveiling the photoluminescence dynamics of gold nanoclusters with fluorescence correlation spectroscopy.

Gold nanoclusters (AuNCs) have captured significant interest for their photoluminescent properties; however, their rapid photodynamics remain elusive while probed by ensemble-averaging spectroscopy techniques. To address this challenge, we use fluorescence correlation spectroscopy (FCS) to uncover the photoluminescence dynamics of colloidal Au18(SG)14 nanoclusters. Our FCS analysis reveals the photoluminescence (PL) brightness per nanocluster, elucidating the impact of photoexcitation saturation and ligand interactions. Unlike DNA-encapsulated silver nanoclusters, their gold counterparts notably exhibit minimal blinking, with moderate amplitudes and 200 µs characteristic times. Our data also clearly reveal the occurrence of photon antibunching in the PL emission, showcasing the quantum nature of the PL process, with each AuNC acting as an individual quantum source. Using zero-mode waveguide nanoapertures, we achieve a 16-fold enhancement of the PL brightness of individual AuNCs. This constitutes an important enabling proof-of-concept for tailoring emission properties through nanophotonics. Overall, our study bridges the gap between ensemble-averaged techniques and single-molecule spectroscopy, offering new insights into AuNC photodynamics for biosensing and imaging applications.