Dynamic structure mediates halophilic adaptation of a DNA polymerase from the deep-sea brines of the Red Sea.

The deep-sea brines of the Red Sea are remote and unexplored environments characterized by high temperatures, anoxic water, and elevated concentrations of salt and heavy metals. This environment provides a rare system to study the interplay between halophilic and thermophilic adaptation in biologic macromolecules. The present article reports the first DNA polymerase with halophilic and thermophilic features. Biochemical and structural analysis by Raman and circular dichroism spectroscopy showed that the charge distribution on the protein's surface mediates the structural balance between stability for thermal adaptation and flexibility for counteracting the salt-induced rigid and nonfunctional hydrophobic packing. Salt bridge interactions via increased negative and positive charges contribute to structural stability. Salt tolerance, conversely, is mediated by a dynamic structure that becomes more fixed and functional with increasing salt concentration. We propose that repulsive forces among excess negative charges, in addition to a high percentage of negatively charged random coils, mediate this structural dynamism. This knowledge enabled us to engineer a halophilic version of Thermococcus kodakarensis DNA polymerase.-Takahashi, M., Takahashi, E., Joudeh, L. I., Marini, M., Das, G., Elshenawy, M. M., Akal, A., Sakashita, K., Alam, I., Tehseen, M., Sobhy, M. A., Stingl, U., Merzaban, J. S., Di Fabrizio, E., Hamdan, S. M. Dynamic structure mediates halophilic adaptation of a DNA polymerase from the deep-sea brines of the Red Sea.

Microfluidic device for continuous single cells analysis via Raman spectroscopy enhanced by integrated plasmonic nanodimers.

In this work a Raman flow cytometer is presented. It consists of a microfluidic device that takes advantages of the basic principles of Raman spectroscopy and flow cytometry. The microfluidic device integrates calibrated microfluidic channels- where the cells can flow one-by-one -, allowing single cell Raman analysis. The microfluidic channel integrates plasmonic nanodimers in a fluidic trapping region. In this way it is possible to perform Enhanced Raman Spectroscopy on single cell. These allow a label-free analysis, providing information about the biochemical content of membrane and cytoplasm of the each cell. Experiments are performed on red blood cells (RBCs), peripheral blood lymphocytes (PBLs) and myelogenous leukemia tumor cells (K562).

Direct determination of the resonance properties of metallic conical nanoantennas.

We present a simple method that is able to predict the resonant frequencies of a metallic conical nanoantenna. The calculation is based on an integral relation that takes into account the dependence of the effective refractive index of the plasmonic mode on the cone radius. Numerical simulations retrieving the near field properties of nanocones with different lengths are also performed for comparison. The fine agreement between the two approaches demonstrates the validity of our method.

Multifunctional substrates of thin porous alumina for cell biosensors.

We have fabricated anodic porous alumina from thin films (100/500 nm) of aluminium deposited on technological substrates of silicon/glass, and investigated the feasibility of this material as a surface for the development of analytical biosensors aiming to assess the status of living cells. To this goal, porous alumina surfaces with fixed pitch and variable pore size were analyzed for various functionalities. Gold coated (about 25 nm) alumina revealed surface enhanced Raman scattering increasing with the decrease in wall thickness, with factor up to values of approximately 10(4) with respect to the flat gold surface. Bare porous alumina was employed for micro-patterning and observation via fluorescence images of dye molecules, which demonstrated the surface capability for a drug-loading device. NIH-3T3 fibroblast cells were cultured in vitro and examined after 2 days since seeding, and no significant (P > 0.05) differences in their proliferation were observed on porous and non-porous materials. The effect on cell cultures of pore size in the range of 50-130 nm--with pore pitch of about 250 nm--showed no significant differences in cell viability and similar levels in all cases as on a control substrate. Future work will address combination of all above capabilities into a single device.

3D hollow nanostructures as building blocks for multifunctional plasmonics.

We present an advanced and robust technology to realize 3D hollow plasmonic nanostructures which are tunable in size, shape, and layout. The presented architectures offer new and unconventional properties such as the realization of 3D plasmonic hollow nanocavities with high electric field confinement and enhancement, finely structured extinction profiles, and broad band optical absorption. The 3D nature of the devices can overcome intrinsic difficulties related to conventional architectures in a wide range of multidisciplinary applications.

Interplay between electric and magnetic effect in adiabatic polaritonic systems.

We report on the possibility of realizing adiabatic compression of polaritonic wave on a metallic conical nano-structure through an oscillating electric potential (quasi dynamic regime). By comparing this result with an electromagnetic wave excitation, we were able to relate the classical lighting-rod effect to adiabatic compression. Furthermore, we show that while the magnetic contribution plays a marginal role in the formation of adiabatic compression, it provides a blue shift in the spectral region. In particular, magnetic permeability can be used as a free parameter for tuning the polaritonic resonances. The peculiar form of adiabatic compression is instead dictated by both the source and the metal permittivity. The analysis is performed by starting from a simple electrostatic system to end with the complete electromagnetic one through intermediate situations such as the quasi-electrostatic and quasi-dynamic regimes. Each configuration is defined by a particular set of equations which allows to clearly determine the individual role played by the electric and magnetic contribution in the generation of adiabatic compression. We notice that these findings can be applied for the realization of a THz nano-metric generator.

Carbon dioxide adsorbents from flame-made diesel soot nanoparticles.

Carbon dioxide (CO2) is the top contributor to global warming. On the other, soot particles formed during fuel combustion and released into the atmosphere are harmful and also contribute to global warming. It would therefore be highly advantageous to capture soot and make use of it as a feedstock to synthesize carbon-based materials for applications such as carbon dioxide adsorption. In this work, flame-made diesel soot nanoparticles were used to produce a variety of activated carbons by combined oxidative treatment with hydrogen peroxide (H2O2) and potassium hydroxide (KOH), and their performance towards CO2 adsorption was evaluated. The effect of the chemical activation of soot with H2O2 for different reaction times and with KOH on the physicochemical properties of the activated carbons was investigated and compared to fresh soot. Interestingly, hollow aggregates of carbonaceous nanoparticles of a high interplanar distance, reduced polycyclic aromatic hydrocarbons (PAH) size, shorter PAH stacks, mesoporous structure, and a high content of oxygen functionalities along with other structural defects in PAHs were obtained in the synthesized activated carbons. Among the various analysis techniques employed, Raman spectroscopy indicated that the ID/IG ratio in soot decreased after simultaneous chemical treatment, though it did not indicate any enhancement in the graphitic character since the carbonyl and carboxylic containing PAHs and monovacancies (which cause defects in PAHs) also contribute to the increase in the intensity of the graphitic band. The activated carbons possessed promising CO2 adsorption capacities, adsorption kinetics and CO2/N2 selectivity. For example, one of the activated carbons, following H2O2 treatment for 9 h and a subsequent KOH activation, exhibited a CO2 adsorption capacity of 1.78 mmol/g at 1 bar and 25 °C, representing an increase of 161 % in capacity as compared to fresh soot. Hollow aggregates of carbonaceous nanoparticles consisting of shorter PAHs with a larger number of defects led to enhanced CO2 adsorption rate and CO2/N2 selectivity on activated carbons.

Surface plasmon polariton compression through radially and linearly polarized source.

We report on the possibility of realizing a radial mode on a metallic conical structure by means of a linearly polarized incident wave. This result is utilized for observing surface plasmon polaritons adiabatic compression on a tapered conical nanostructure. The ingredients for radial mode generation are described in terms of phase-matching of the components of the electromagnetic field. We conclude by showing the robustness of this approach, explaining the polaritonic behavior as a function of the device geometry.

Fabrication of large-area ordered and reproducible nanostructures for SERS biosensor application.

We propose a large-area SERS device with efficient fluorescence quenching capability. The substrate is based on anodic porous alumina templates with various pore size and wall thickness as small as 15 and 36 nm, respectively. The nano-patterned SERS substrate, with excellent control and reproducibility of plasmon-polaritons generation, shows very efficient enhanced Raman signal in the presence of intrinsically fluorescent molecules such as cresyl violet, rhodamine, and green fluorescent protein. This work demonstrates that, when the nanostructures are properly designed and fabricated, Raman and fluorescence spectroscopy can be used in combination in order to obtain complementary molecular informations. Theoretical simulation shows excellent agreement with the experimental findings. The enhancement factor is found to be 10(3)-10(4), with respect to flat gold surface when the molecules are supposed to be closely packed, with considerable fluorescence suppression, showing a promising disposable biosensor.

Combining Azimuthal and Polar Angle Resolved Shadow Mask Deposition and Nanosphere Lithography to Uncover Unique Nano-Crystals.

In this article, we present a systematic investigation on a multistep nanosphere lithography technique to uncover its potential in fabricating a wide range of two- and three-dimensional nanostructures. A tilted (polar angle) electron beam shower on a nanosphere mask results in an angled shadow mask deposition. The shape of the shadow also depends on the azimuthal angle of the mask sitting on top of the substrate. We performed angled shadow mask depositions with systematic variation of these two angular parameters, giving rise to complex nanostructures (down to 50 nm), repeated over a large area without defect. In this article, nanosphere lithography with two- and four-fold azimuthal symmetry was studied at constant tilt angles followed by variations in tilt without azimuthal rotation of the substrate. Finally, both angular parameters were simultaneously varied. The structure of shadow crystals was explained using Matlab simulation. This work stretches the horizons of nanosphere lithography, opening up new scopes in plasmonic and magnonic research.

Correlative Raman-Electron-Light (CREL) Microscopy Analysis of Lipid Droplets in Melanoma Cancer Stem Cells.

Among all neoplasms, melanoma is characterized by a very high percentage of cancer stem cells (CSCs). Several markers have been proposed for their identification, and lipid droplets (LDs) are among them. Different techniques are used for their characterization such as mass spectrometry, imaging techniques, and vibrational spectroscopies. Some emerging experimental approaches for the study of LDs are represented by correlative light-electron microscopy and by correlative Raman imaging-scanning electron microscopy (SEM). Based on these scientific approaches, we developed a novel methodology (CREL) by combining Raman micro-spectroscopy, confocal fluorescence microscopy, and SEM coupled with an energy-dispersive X-ray spectroscopy module. This procedure correlated cellular morphology, chemical properties, and spatial distribution from the same region of interest, and in this work, we presented the application of CREL for the analysis of LDs within patient-derived melanoma CSCs (MCSCs).

Extremely large extinction efficiency and field enhancement in terahertz resonant dipole nanoantennas.

The distinctive ability of nanometallic structures to manipulate light at the nanoscale has recently promoted their use for a spectacular set of applications in a wide range of areas of research including artificial optical materials, nano-imaging, biosensing, and nonlinear optics. Here we transfer this concept to the terahertz spectral region, demonstrating a metal nanostructure in shape of a dipole nanoantenna, which can efficiently resonate at terahertz frequencies, showing an effective cross section >100 times larger than its geometrical area, and a field enhancement factor of ~280, confined on a lateral section of ~λ/1,000. These results lead to immediate applications in terahertz artificial materials exhibiting giant dichroism, suggest the use of dipole nanoantennas in nanostructure-based terahertz metamaterials, and pave the way for nanoantenna-enhanced terahertz few-molecule spectroscopy and localized terahertz nonlinear optics.

Nanoparticle microinjection and Raman spectroscopy as tools for nanotoxicology studies.

Microinjection techniques and Raman spectroscopy have been combined to provide a new methodology to investigate the cytotoxic effects due to the interaction of nanomaterials with cells. In the present work, this novel technique has been used to investigate the effects of Ag and Fe(3)O(4) nanoparticles on Hela cells. The nanoparticles are microinjected inside the cells and these latter ones are probed by means of Raman spectroscopy after a short incubation time, in order to highlight the first and impulsive mechanisms developed by the cells to counteract the presence of the nanoparticles. The results put in evidence a different behaviour of the cells treated with nanoparticles in comparison with the control cells; these differences are supposed to be generated by an emerging oxidative stress due to the nanoparticles. The achieved results demonstrate the suitability of the proposed method as a new tool for nanotoxicity studies.

Tip enhanced Raman scattering with adiabatic plasmon focusing tips.

Tip-Enhanced Raman spectroscopy (TERS) is a promising microscopy technique which combines, in principle, outstanding spatial resolution with a detailed chemical analysis of the sample. However, as yet, it is not routinely used although an increasing number of research groups are becoming more actively involved in the field. Among the several reasons which can explain the relatively low usage of TERS, the lack of reproducibility of tips as field enhancers is probably the most critical. Here we propose and demonstrate a TERS microscope which uses photonic engineered tips. These tips are based on standard silicon nitride atomic force microscope (AFM) cantilevers. A photonic crystal together with a plasmonic waveguide focuses the Raman excitation laser to the apex of the waveguide, enabling a photon confinement equivalent to the radius of curvature of the nanofabricated tip. These tips were successfully applied here in both AFM imaging and high resolution Raman spectroscopy. The new tips produced AFM imaging performances comparable with the best AFM commercial tips. Moreover, we demonstrate that the photonic crystal combined with the plasmonic waveguide acts effectively as a localized near field emitter.

Clustering of Major Histocompatibility Complex-Class I Molecules in Healthy and Cancer Colon Cells Revealed from Their Nanomechanical Properties.

The activation of the T cell mediated immune response relies on the fine interaction between the T cell receptor on the immune cell and the antigen-presenting major histocompatibility complex (MHC) molecules on the membrane surface of antigen-presenting cells. Both the distribution and quantity of MHC/peptide complexes and their adequate morphological presentation affect the activation of the immune cells. In several types of cancer the immune response is down-regulated due to the low expression of MHC-class I (MHC-I) molecules on the cell's surface, and in addition, the mechanical properties of the membrane seem to play a role. Herein, we investigate the distribution of MHC-I molecules and the related nanoscale mechanical environment on the cell surface of two cell lines derived from colon adenocarcinoma and a healthy epithelial colon reference cell line. Atomic force microscopy (AFM) force spectroscopy analysis using an antibody-tagged pyramidal probe specific for MHC-I molecules and a formula that relates the elasticity of the cell to the energy of adhesion revealed the different population distributions of MHC-I molecules in healthy cells compared to cancer cells. We found that MHC-I molecules are significantly less expressed in cancer cells. Moreover, the local elastic modulus is significantly reduced in cancer cells. We speculate that these results might be related to the proven ability of cancer cells to evade the immune system, not only by reducing MHC-I cell surface expression but also by modifying the local mechanical properties affecting the overall morphology of MHC-I synapse presentation to immune cells.

Phonon Dynamics and Transport Properties of Copper Thiocyanate and Copper Selenocyanate Pseudohalides.

The phonon transport properties of CuSCN and CuSeCN have been investigated using the density functional theory and semiclassical Boltzmann transport theory. The Perdew-Burke-Ernzerhof functional shows an indirect (direct) electronic band gap of 2.18 eV (1.80 eV) for CuSCN (CuSeCN). The calculated phonon band structure shows that both compounds are dynamically stable. The Debye temperature of the acoustic phonons is 122 and 107 K for CuSCN and CuSeCN, respectively. The extended in-plane bond lengths as compared to the out-of-plane bond lengths result in phonon softening and hence, low lattice thermal conductivity. The calculated room temperature in-plane (out-of-plane) lattice thermal conductivity of CuSCN and CuSeCN is 2.39 W/mK (4.51 W/mK) and 1.70 W/mK (3.83 W/mK), respectively. The high phonon scattering rates in CuSeCN give rise to in-plane low lattice thermal conductivities. The room-temperature Grüneisen parameters of CuSCN and CuSeCN are found to be 0.98 and 1.08, respectively.

A droplet reactor on a super-hydrophobic surface allows control and characterization of amyloid fibril growth.

Methods to produce protein amyloid fibrils, in vitro, and in situ structure characterization, are of primary importance in biology, medicine, and pharmacology. We first demonstrated the droplet on a super-hydrophobic substrate as the reactor to produce protein amyloid fibrils with real-time monitoring of the growth process by using combined light-sheet microscopy and thermal imaging. The molecular structures were characterized by Raman spectroscopy, X-ray diffraction and X-ray scattering. We demonstrated that the convective flow induced by the temperature gradient of the sample is the main driving force in the growth of well-ordered protein fibrils. Particular attention was devoted to PHF6 peptide and full-length Tau441 protein to form amyloid fibrils. By a combined experimental with the molecular dynamics simulations, the conformational polymorphism of these amyloid fibrils were characterized. The study provided a feasible procedure to optimize the amyloid fibrils formation and characterizations of other types of proteins in future studies.

Waveguiding and SERS Simplified Raman Spectroscopy on Biological Samples.

Biomarkers detection at an ultra-low concentration in biofluids (blood, serum, saliva, etc.) is a key point for the early diagnosis success and the development of personalized therapies. However, it remains a challenge due to limiting factors like (i) the complexity of analyzed media, and (ii) the aspecificity detection and the poor sensitivity of the conventional methods. In addition, several applications require the integration of the primary sensors with other devices (microfluidic devices, capillaries, flasks, vials, etc.) where transducing the signal might be difficult, reducing performances and applicability. In the present work, we demonstrate a new class of optical biosensor we have developed integrating an optical waveguide (OWG) with specific plasmonic surfaces. Exploiting the plasmonic resonance, the devices give consistent results in surface enhanced Raman spectroscopy (SERS) for continuous and label-free detection of biological compounds. The OWG allows driving optical signals in the proximity of SERS surfaces (detection area) overcoming spatial constraints, in order to reach places previously optically inaccessible. A rutile prism couples the remote laser source to the OWG, while a Raman spectrometer collects the SERS far field scattering. The present biosensors were implemented by a simple fabrication process, which includes photolithography and nanofabrication. By using such devices, it was possible to detect cell metabolites like Phenylalanine (Phe), Adenosine 5-triphosphate sodium hydrate (ATP), Sodium Lactate, Human Interleukin 6 (IL6), and relate them to possible metabolic pathway variation.

Plasmonic Nanowires for Wide Wavelength Range Molecular Sensing.

In this paper, we propose the use of a standing nanowires array, constituted by plasmonic active gold wires grown on iron disks, and partially immersed in a supporting alumina matrix, for surface-enhanced Raman spectroscopy applications. The galvanic process was used to fabricate nanowires in pores of anodized alumina template, making this device cost-effective. This fabrication method allows for the selection of size, diameter, and spatial arrangement of nanowires. The proposed device, thanks to a detailed design analysis, demonstrates a broadband plasmonic enhancement effect useful for many standard excitation wavelengths in the visible and NIR. The trigonal pores arrangement gives an efficiency weakly dependent on polarization. The devices, tested with 633 and 830 nm laser lines, show a significant Raman enhancement factor, up to around 6 × 104, with respect to the flat gold surface, used as a reference for the measurements of the investigated molecules.

Plasmonic Color-Graded Nanosystems with Achromatic Subwavelength Architectures for Light Filtering and Advanced SERS Detection.

Plasmonic color-graded systems are devices featuring a spatially variable plasmonic response over their surface. They are widely used as nanoscale color filters; their typical size is small enough to allow integration with miniaturized electronic circuits, paving the way to realize novel nanophotonic devices. Currently, most plasmonic color-graded systems are intrinsically discrete because their chromatic response exploits the tailored plasmon resonance of microarchitectures characterized by different size or geometry for each target color. Here, we report the realization of multifunctional plasmon-graded devices where continuously graded chromatic response is achieved by smoothly tuning the composition of the resonator material while simultaneously maintaining an achromatic nanoscale geometry. The result is a new class of versatile materials: we show their application as plasmonic filters with a potential pixel size smaller than half of the exciting wavelength but also as multiplexed surface-enhanced Raman spectroscopy (SERS) substrates. Many more implementations, such as photovoltaic efficiency boosters or color routers, await and will benefit from the low fabrication cost and intrinsic plasmonic flexibility of the presented systems.