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PR65 is the HEAT repeat scaffold subunit of the heterotrimeric protein phosphatase 2A (PP2A) and an archetypal tandem repeat protein. Its conformational mechanics plays a crucial role in PP2A function by opening/closing substrate binding/catalysis interface. Using in silico saturation mutagenesis, we identified PR65 "hinge" residues whose substitutions could alter its conformational adaptability and thereby PP2A function, and selected six mutations that were verified to be expressed and soluble. Molecular simulations and nanoaperture optical tweezers revealed consistent results on the specific effects of the mutations on the structure and dynamics of PR65. Two mutants observed in simulations to stabilize extended/open conformations exhibited higher corner frequencies and lower translational scattering in experiments, indicating a shift toward extended conformations, whereas another displayed the opposite features, confirmed by both simulations and experiments. The study highlights the power of single-molecule nanoaperture-based tweezers integrated with in silico approaches for exploring the effect of mutations on protein structure and dynamics.
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Simulação de Dinâmica Molecular , Pinças Ópticas , Mutação Puntual , Conformação Proteica , Proteína Fosfatase 2/genética , Proteína Fosfatase 2/química , Proteína Fosfatase 2/metabolismo , HumanosRESUMO
Temperature changes in plasmonic traps can affect biomolecules and quantum emitters; therefore, several works have sought out the capability of measuring the local temperature. Those works used ionic nanopore currents, fluorescence emission variations, and fluorescence-based diffusion tracking to measure the temperature dependence of shaped nanoapertures in metal films. Here, we make use of a stable erbium-containing NaYF4 nanocrystal that gives local temperature dependence while trapped in the nanoaperture hot spot. Ratiometric analysis of the emission at different wavelengths gives local temperature variation. Since the gold film dominates the thermal characteristic, we find that films of thickness 70, 100, and 130 nm give 0.64, 0.37, and 0.25 K/mW temperature change with laser power. Therefore, using thicker films can be effective in reducing the heating when it is not desired.
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Single unmodified biomolecules in solution can be observed and characterized by interferometric imaging approaches; however, Rayleigh scattering limits this to larger proteins (typically >30 kDa). We observe real-time image tracking of unmodified proteins down to 14 kDa using interference imaging enhanced by surface plasmons launched at an aperture in a metal film. The larger proteins show slower diffusion, quantified by tracking. When the diffusing protein is finally trapped by the nanoaperture, we perform complementary power spectral density and noise amplitude analysis, which gives information about the protein. This approach allows for rapid protein characterization with minimal sample preparation and opens the door to characterizing protein interactions in real time.
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PR65 is the HEAT-repeat scaffold subunit of the heterotrimeric protein phosphatase 2A (PP2A) and an archetypal tandem-repeat protein, forming a spring-like architecture. PR65 conformational mechanics play a crucial role in PP2A function by opening/closing the substrate-binding/catalysis interface. Using in-silico saturation mutagenesis we identified "hinge" residues of PR65, whose substitutions are predicted to restrict its conformational adaptability and thereby disrupt PP2A function. Molecular simulations revealed that a subset of hinge mutations stabilized the extended/open conformation, whereas another had the opposite effect. By trapping in nanoaperture optical tweezer, we characterized PR65 motion and showed that the former mutants exhibited higher corner frequencies and lower translational scattering, indicating a shift towards extended conformations, whereas the latter showed the opposite behavior. Thus, experiments confirm the conformations predicted computationally. The study highlights the utility of nanoaperture-based tweezers for exploring structure and dynamics, and the power of integrating this single-molecule method with in silico approaches.
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PR65, a horseshoe-shaped scaffold composed of 15 HEAT (observed in Huntingtin, elongation factor 3, protein phosphatase 2A, and the yeast kinase TOR1) repeats, forms, together with catalytic and regulatory subunits, the heterotrimeric protein phosphatase PP2A. We examined the role of PR65 in enabling PP2A enzymatic activity with computations at various levels of complexity, including hybrid approaches that combine full-atomic and elastic network models. Our study points to the high flexibility of this scaffold allowing for end-to-end distance fluctuations of 40-50 Å between compact and extended conformations. Notably, the intrinsic dynamics of PR65 facilitates complexation with the catalytic subunit and is retained in the PP2A complex enabling PR65 to engage the two domains of the catalytic subunit and provide the mechanical framework for enzymatic activity, with support from the regulatory subunit. In particular, the intra-repeat coils at the C-terminal arm play an important role in allosterically mediating the collective dynamics of PP2A, pointing to target sites for modulating PR65 function.
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Proteína Fosfatase 2 , Proteína Fosfatase 2/genética , Regulação Alostérica , Ligação Proteica , Domínio CatalíticoRESUMO
Single molecule analysis of proteins in an aqueous environment without modification (e.g., labels or tethers) elucidates their biophysics and interactions relevant to drug discovery. By combining fringe-field dielectrophoresis with nanoaperture optical tweezers we demonstrate an order of magnitude faster time-to-trap for proteins when the counter electrode is outside of the solution. When the counter electrode is inside the solution (the more common configuration found in the literature), electrophoresis speeds up the trapping of polystyrene nanospheres, but this was not effective for proteins in general. Since time-to-trap is critical for high-thoughput analysis, these findings are a major advancement to the nanoaperture optical trapping technique for protein analysis.
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Pinças Ópticas , Proteínas , Biofísica , Eletroforese , PoliestirenosRESUMO
We demonstrate reflection geometry optical trapping using double nanoholes in a metal film. Symmetry breaking of the double nanohole allows for selecting the scattered trapping laser light of orthogonal polarization to the incident beam. This orthogonal polarization light shows a few percent increase when the nanoparticle (e.g., a 20 nm polystyrene particle, or protein bovine serum albumin) is trapped. The reflection geometry simplifies the optical setup and frees up one side of the trap, which has great potential for adding microfluidics to the other side or working with opaque or highly scattering samples.
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A dielectric (nano)particle can influence the local electromagnetic field and thereby alter its interaction with that field through the process of self-induced back-action. While this phenomenon is usually considered theoretically as a change in a cavity resonance frequency, such theoretical approaches are not as appropriate when considering systems away from resonance, such as with a subwavelength aperture in a metal film. Here we consider the interaction between an aperture, modelled with Bethe theory as a magnetic dipole, and a Rayleigh particle, modelled as an electric dipole. Using this magnetic dipole - electric dipole interaction, we quantify the self-induced back-action of the particle on the aperture transmission and the optical trapping potential. The model shows quantitative agreement with finite-difference time-domain simulations. This shows that the physics of self-induced back-action for an aperture and a nanoparticle can be understood in terms of dipole-dipole coupling.
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Perovskite quantum dots (PQDs) provide a robust solution-based approach to efficient solar cells, bright light emitting devices, and quantum sources of light. Quantifying heterogeneity and understanding coupling between dots is critical for these applications. We use double-nanohole optical trapping to size individual dots and correlate to emission energy shifts from quantum confinement. We were able to assemble a second dot in the trap, which allows us to observe the coupling between dots. We observe a systematic red-shift of 1.1 ± 0.6 meV in the emission wavelength. Theoretical analysis shows that the observed shift is consistent with resonant energy transfer and is unusually large due to moderate-to-large quantum confinement in PQDs. This demonstrates the promise of PQDs for entanglement in quantum information applications. This work enables future in situ control of PQD growth as well as studies of the coupling between small PQD assemblies with quantum information applications in mind.
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Nanohole optical tweezers have been used by several groups to trap and analyze proteins. In this work, we demonstrate that it is possible to create high-performance double nanohole (DNH) substrates for trapping proteins without the need for any top-down approaches (such as electron microscopy or focused-ion beam milling). Using polarization analysis, we identify DNHs as well as determine their orientation and then use them for trapping. We are also able to identify other hole configurations, such as single, trimers and other clusters. We explore changing the substrate from glass to polyvinyl chloride to enhance trapping ability, showing 7 times lower minimum trapping power, which we believe is due to reduced surface repulsion. Finally, we present tape exfoliation as a means to expose DNHs without damaging sonication or chemical methods. Overall, these approaches make high quality optical trapping using DNH structures accessible to a broad scientific community.
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Two independent groups designed nanoantennas for detecting mid-infrared light.
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TemperaturaRESUMO
We present a fully analytic theory to study the power and field enhancement inside a real metal slit. A generalized formula for the reflection coefficient at the interface of the slit is derived. The resulting expression is purely analytic and the reflection coefficient can be simply evaluated to provide physical insight, while not requiring complicated numerical simulations. The calculated values of reflection phase and amplitude are then used in the Fabry-Pérot formalism to compute the electric field and the power inside the slit. It is shown that the power attains its maximum value when the scattering and the absorption cross-sections of the slit are equal, a confirmation of the maximum power transfer theorem for this case. The analytic results agree well with numerical simulations, which is promising for optimizing performance in applications ranging from modulators to optical tweezers.
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Single-photon sources are required for quantum technologies and can be created from individual atoms and atom-like defects. Erbium ions produce single photons at low-loss fiber optic wavelengths, but they have low emission rates, making them challenging to isolate reliably. Here, we tune the size of gold double nanoholes (DNHs) to enhance the emission of single erbium emitters, achieving 50× enhancement over rectangular apertures previously demonstrated. This produces enough enhancement to show emission from single nanocrystals at wavelengths not seen in our previous work, i.e., 400 and 1550 nm. We observe discrete levels of emission for nanocrystals with low numbers of emitters and demonstrate isolating single emitters. We describe how the trapping time is proportional to the enhancement factor for a given DNH structure, giving us an independent way to measure the enhancement. This shows a promising path to achieving single emitter sources at 1550 nm.
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Upconverted light from nanostructured metal surfaces can be produced by harmonic generation and multi-photon luminescence; however, these are very weak processes and require extremely high field intensities to produce a measurable signal. Here we report on bright emission, 5 orders of magnitude greater than harmonic generation, that can be seen from metal tunnel junctions that we believe is due to light-induced inelastic tunneling emission. Like inelastic tunneling light emission, which was recently reported to have 2% conversion efficiency per tunneling event, the emission wavelength recorded varies with the local electric field applied; however, here the field is from a 1560 nm femtosecond pulsed laser source. Finite-difference time-domain simulations of the experimental conditions show the local field is sufficient to generate tunneling-based inelastic light emission in the visible regime. This phenomenon is promising for producing ultrafast upconverted light emission with higher efficiency than conventional nonlinear processes.
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Single-photon emitters based on individual atoms or individual atomic-like defects are highly sought-after components for future quantum technologies. A key challenge in this field is how to isolate just one such emitter; the best approaches still have an active emitter yield of only 50% so that deterministic integration of single active emitters is not yet possible. Here, we demonstrate the ability to isolate individual erbium emitters embedded in 20 nm nanocrystals of NaYF4 using plasmonic aperture optical tweezers. The optical tweezers capture the nanocrystal, whereas the plasmonic aperture enhances the emission of the Er and allows the measurement of discrete emission rate values corresponding to different numbers of erbium ions. Three separate synthesis runs show near-Poissonian distribution in the discrete levels of emission yield that correspond to the expected ion concentrations, indicating that the yield of active emitters is approximately 80%. Fortunately, the trap allows for selecting the nanocrystals with only a single emitter, and so this gives a route to isolating and integrating single emitters in a deterministic way. This demonstration is a promising step toward single-photon quantum information technologies that utilize single ions in a solid-state medium, particularly because Er emits in the low-loss fiber-optic 1550 nm telecom band.
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We use metal nanostructures (nanoplasmonics) excited with dual frequency lasers to generate and detect high-frequency (>10 GHz) sound wave resonances in water. The difference frequency between the two lasers causes beating in the intensity, which results in a drop in the transmission through the nanostructure when an acoustic resonance is excited. By observing the resonance frequency shifts with changing nanostructure size, the transition from slow to fast sound in water is inferred, which has been measured by inelastic scattering methods in the past. The observed behavior shows remarkable similarities to finite element simulations using a simple Debye model for sound velocity (without fitting parameters).
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Double-nanoholes fabricated by colloidal lithography were used for trapping single colloidal particles and single proteins. A gap separation of 60 nm between the cusps of the double-nanohole was achieved in a gold film of 70 nm thickness sputter coated onglass. The cusp separation was reduced steadily down to 10 nm by plasma etching the colloidal particles prior to sputter coating. Scanning electron microscopy was used to locate a particular double-nanohole and it was registered for later microscopy experiments. 30 nm polystyrene particles, the rubisco protein and bovine serum albumin were trapped using a laser focused through the aperture. Compared to other methods that require top-down nanofabrication, this approach is inexpensive and produces high-quality samples.
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Técnicas Biossensoriais , Coloides/química , Nanopartículas/química , Nanotecnologia/métodos , Ribulose-Bifosfato Carboxilase/química , Soroalbumina Bovina/química , Ouro , Microscopia Eletrônica de Varredura , Pinças Ópticas , Poliestirenos/químicaRESUMO
We demonstrate coupling and directivity enhancement of electromagnetic fields emerging from a single metallic nanoaperture at the tip of a single-mode optical fiber. We achieve this by using circular grooves flanking the nanoaperture perforated in a 100 nm thick gold film. The film with nanostructure is transferred to the fiber tip by aligned stripping with optical epoxy. When incident from both sides of the nanoaperture, enhancement factors of 2.2 and 2.4 in power coupling into the fiber and in beaming into free-space were obtained. Numerical simulations show that the optimum grating period is nearly identical to the surface plasmon polariton wavelength that can be supported at the gold-epoxy interface. This integrated platform couples light between the single mode fiber and the nanoapeture without the need for cumbersome optics, with applications for optical trapping and single-photon detection.
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The basic theoretical understanding of light interacting with nanostructured metals that has existed since the early 1900s has become more relevant in the last two decades, largely because of new approaches to structure metals down to the nanometer scale or smaller. Here, a broad overview of the concepts and applications of nanostructuring metals for light-based technologies is given. The theory of the response of metals to an applied oscillating field is given, including a discussion of nonlocal, nonlinear and quantum effects. Using this metal response, the guiding of electromagnetic (light) waves using metals is given, with a particular emphasis on the impact of nanostructured metals for tighter confinement and slower propagation. Similarly, the influence of metal nanostructures on light scattering by isolated metal structures, like nanoparticles and nanoantennas, is described, with basic results presented including plasmonic/circuit resonances, the single channel limit, directivity enhancement, the maximum power transfer theorem, limits on the magnetic response from kinetic inductance and the scaling of gap plasmons to the nanometer scale and smaller. A brief overview of nanofabrication approaches to creating metal nanostructures is given. Finally, existing and emerging light-based applications are presented, including those for sensing, spectroscopy (including local refractive index, Raman, IR absorption), detection (including Schottky detectors), switching (including terahertz photoconductive antennas), modulation, energy harvesting and photocatalysis, light emission (including lasers and tunneling based light emission), optical tweezing, nonlinear optics, subwavelength imaging and lithography and high density data storage.