Direct imaging of polymer filaments pulled from rebounding drops.

Polymer filaments form the foundation of biology from cell scaffolding to DNA. Their study and fabrication play an important role in a wide range of processes from tissue engineering to molecular machines. We present a simple method to deposit stretched polymer fibers between micro-pillars. This occurs when a polymeric drop impacts on and rebounds from an inclined superhydrophobic substrate. It wets the top of the pillars and pulls out liquid filaments which are stretched and can attach to adjacent pillars leaving minuscule threads, with the solvent evaporating to leave the exposed polymers. We use high-speed video at the microscale to characterize the most robust filament-forming configurations, by varying the impact velocity, substrate structure and inclination angle, as well as the PEO-polymer concentration. Impacts onto plant leaves or a randomized nano-structured surface leads to the formation of a branched structure, through filament mergers at the free surface of the drop. SEM shows the deposition of filament bundles which are thinner than those formed by evaporation or rolling drops. Raman spectroscopy identifies the native mode B stretched DNA filaments from aqueous-solution droplets.

Modulated nanowire scaffold for highly efficient differentiation of mesenchymal stem cells.

BACKGROUND: Nanotopographical cues play a critical role as drivers of mesenchymal stem cell differentiation. Nanowire scaffolds, in this regard, provide unique and adaptable nanostructured surfaces with focal points for adhesion and with elastic properties determined by nanowire stiffness. RESULTS: We show that a scaffold of nanowires, which are remotely actuated by a magnetic field, mechanically stimulates mesenchymal stem cells. Osteopontin, a marker of osteogenesis onset, was expressed after cells were cultured for 1 week on top of the scaffold. Applying a magnetic field significantly boosted differentiation due to mechanical stimulation of the cells by the active deflection of the nanowire tips. The onset of differentiation was reduced to 2 days of culture based on the upregulation of several osteogenesis markers. Moreover, this was observed in the absence of any external differentiation factors. CONCLUSIONS: The magneto-mechanically modulated nanosurface enhanced the osteogenic differentiation capabilities of mesenchymal stem cells, and it provides a customizable tool for stem cell research and tissue engineering.

Biofuel powered glucose detection in bodily fluids with an n-type conjugated polymer.

A promising class of materials for applications that rely on electron transfer for signal generation are the n-type semiconducting polymers. Here we demonstrate the integration of an n-type conjugated polymer with a redox enzyme for the autonomous detection of glucose and power generation from bodily fluids. The reversible, mediator-free, miniaturized glucose sensor is an enzyme-coupled organic electrochemical transistor with a detection range of six orders of magnitude. This n-type polymer is also used as an anode and paired with a polymeric cathode in an enzymatic fuel cell to convert the chemical energy of glucose and oxygen into electrical power. The all-polymer biofuel cell shows a performance that scales with the glucose content in the solution and a stability that exceeds 30 days. Moreover, at physiologically relevant glucose concentrations and from fluids such as human saliva, it generates enough power to operate an organic electrochemical transistor, thus contributes to the technological advancement of self-powered micrometre-scale sensors and actuators that run on metabolites produced in the body.

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.

The phototransduction machinery in the rod outer segment has a strong efficacy gradient.

Rod photoreceptors consist of an outer segment (OS) and an inner segment. Inside the OS a biochemical machinery transforms the rhodopsin photoisomerization into electrical signal. This machinery has been treated as and is thought to be homogenous with marginal inhomogeneities. To verify this assumption, we developed a methodology based on special tapered optical fibers (TOFs) to deliver highly localized light stimulations. By using these TOFs, specific regions of the rod OS could be stimulated with spots of light highly confined in space. As the TOF is moved from the OS base toward its tip, the amplitude of saturating and single photon responses decreases, demonstrating that the efficacy of the transduction machinery is not uniform and is 5-10 times higher at the base than at the tip. This gradient of efficacy of the transduction machinery is attributed to a progressive depletion of the phosphodiesterase along the rod OS. Moreover we demonstrate that, using restricted spots of light, the duration of the photoresponse along the OS does not increase linearly with the light intensity as with diffuse light.

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).

Lipid droplets: a new player in colorectal cancer stem cells unveiled by spectroscopic imaging.

The cancer stem cell (CSC) model is describing tumors as a hierarchical organized system and CSCs are suggested to be responsible for cancer recurrence after therapy. The identification of specific markers of CSCs is therefore of paramount importance. Here, we show that high levels of lipid droplets (LDs) are a distinctive mark of CSCs in colorectal (CR) cancer. This increased lipid content was clearly revealed by label-free Raman spectroscopy and it directly correlates with well-accepted CR-CSC markers as CD133 and Wnt pathway activity. By xenotransplantation experiments, we have finally demonstrated that CR-CSCs overexpressing LDs retain most tumorigenic potential. A relevant conceptual advance in this work is the demonstration that a cellular organelle, the LD, is a signature of CSCs, in addition to molecular markers. A further functional characterization of LDs could lead soon to design new target therapies against CR-CSCs.

Aliphatic peptides show similar self-assembly to amyloid core sequences, challenging the importance of aromatic interactions in amyloidosis.

The self-assembly of abnormally folded proteins into amyloid fibrils is a hallmark of many debilitating diseases, from Alzheimer's and Parkinson diseases to prion-related disorders and diabetes type II. However, the fundamental mechanism of amyloid aggregation remains poorly understood. Core sequences of four to seven amino acids within natural amyloid proteins that form toxic fibrils have been used to study amyloidogenesis. We recently reported a class of systematically designed ultrasmall peptides that self-assemble in water into cross-ß-type fibers. Here we compare the self-assembly of these peptides with natural core sequences. These include core segments from Alzheimer's amyloid-ß, human amylin, and calcitonin. We analyzed the self-assembly process using circular dichroism, electron microscopy, X-ray diffraction, rheology, and molecular dynamics simulations. We found that the designed aliphatic peptides exhibited a similar self-assembly mechanism to several natural sequences, with formation of α-helical intermediates being a common feature. Interestingly, the self-assembly of a second core sequence from amyloid-ß, containing the diphenylalanine motif, was distinctly different from all other examined sequences. The diphenylalanine-containing sequence formed ß-sheet aggregates without going through the α-helical intermediate step, giving a unique fiber-diffraction pattern and simulation structure. Based on these results, we propose a simplified aliphatic model system to study amyloidosis. Our results provide vital insight into the nature of early intermediates formed and suggest that aromatic interactions are not as important in amyloid formation as previously postulated. This information is necessary for developing therapeutic drugs that inhibit and control amyloid formation.

Squeezing terahertz light into nanovolumes: nanoantenna enhanced terahertz spectroscopy (NETS) of semiconductor quantum dots.

Terahertz spectroscopy has vast potentialities in sensing a broad range of elementary excitations (e.g., collective vibrations of molecules, phonons, excitons, etc.). However, the large wavelength associated with terahertz radiation (about 300 µm at 1 THz) severely hinders its interaction with nano-objects, such as nanoparticles, nanorods, nanotubes, and large molecules of biological relevance, practically limiting terahertz studies to macroscopic ensembles of these compounds, in the form of thick pellets of crystallized molecules or highly concentrated solutions of nanomaterials. Here we show that chains of terahertz dipole nanoantennas spaced by nanogaps of 20 nm allow retrieving the spectroscopic signature of a monolayer of cadmium selenide quantum dots, a significant portion of the signal arising from the dots located within the antenna nanocavities. A Fano-like interference between the fundamental antenna mode and the phonon resonance of the quantum dots is observed, accompanied by an absorption enhancement factor greater than one million. NETS can find immediate applications in terahertz spectroscopic studies of nanocrystals and molecules at extremely low concentrations. Furthermore, it shows a practicable route toward the characterization of individual nano-objects at these frequencies.

Writing and functionalisation of suspended DNA nanowires on superhydrophobic pillar arrays.

Nanowire arrays and networks with precisely controlled patterns are very interesting for innovative device concepts in mesoscopic physics. In particular, DNA templates have proven to be versatile for the fabrication of complex structures that obtained functionality via combinations with other materials, for example by functionalisation with molecules or nanoparticles, or by coating with metals. Here, the controlled motion of the a three-phase contact line (TCL) of DNA-loaded drops on superhydrophobic substrates is used to fabricate suspended nanowire arrays. In particular, the deposition of DNA wires is imaged in situ, and different patterns are obtained on hexagonal pillar arrays by controlling the TCL velocity and direction. Robust conductive wires and networks are achieved by coating the wires with a thin layer of gold, and as proof of concept conductivity measurements are performed on single suspended wires. The plastic material of the superhydrophobic pillars ensures electrical isolation from the substrate. The more general versatility of these suspended nanowire networks as functional templates is outlined by fabricating hybrid organic-metal-semiconductor nanowires by growing ZnO nanocrystals onto the metal-coated nanowires.

Human NK cells selective targeting of colon cancer-initiating cells: a role for natural cytotoxicity receptors and MHC class I molecules.

Tumor cell populations have been recently proposed to be composed of two compartments: tumor-initiating cells characterized by a slow and asymmetrical growth, and the "differentiated" cancer cells with a fast and symmetrical growth. Cancer stem cells or cancer-initiating cells (CICs) play a crucial role in tumor recurrence. The resistance of CICs to drugs and irradiation often allows them to survive traditional therapy. NK cells are potent cytotoxic lymphocytes that can recognize tumor cells. In this study, we have analyzed the NK cell recognition of tumor target cells derived from the two cancer cell compartments of colon adenocarcinoma lesions. Our data demonstrate that freshly purified allogeneic NK cells can recognize and kill colorectal carcinoma-derived CICs whereas the non-CIC counterpart of the tumors (differentiated tumor cells), either autologous or allogeneic, is less susceptible to NK cells. This difference in the NK cell susceptibility correlates with higher expression on CICs of ligands for NKp30 and NKp44 in the natural cytotoxicity receptor (NCR) group of activating NK receptors. In contrast, CICs express lower levels of MHC class I, known to inhibit NK recognition, on their surface than do the "differentiated" tumor cells. These data have been validated by confocal microscopy where NCR ligands and MHC class I molecule membrane distribution have been analyzed. Moreover, NK cell receptor blockade in cytotoxicity assays demonstrates that NCRs play a major role in the recognition of CIC targets. This study strengthens the idea that biology-based therapy harnessing NK cells could be an attractive opportunity in solid tumors.

Folic acid functionalized surface highlights 5-methylcytosine-genomic content within circulating tumor cells.

Although the detection of methylated cell free DNA represents one of the most promising approaches for relapse risk assessment in cancer patients, the low concentration of cell-free circulating DNA constitutes the biggest obstacle in the development of DNA methylation-based biomarkers from blood. This paper describes a method for the measurement of genomic methylation content directly on circulating tumor cells (CTC), which could be used to deceive the aforementioned problem. Since CTC are disease related blood-based biomarkers, they result essential to monitor tumor's stadiation, therapy, and early relapsing lesions. Within surface's bio-functionalization and cell's isolation procedure standardization, the presented approach reveals a singular ability to detect high 5-methylcytosine CTC-subset content in the whole CTC compound, by choosing folic acid (FA) as transducer molecule. Sensitivity and specificity, calculated for FA functionalized surface (FA-surface), result respectively on about 83% and 60%. FA-surface, allowing the detection and characterization of early metastatic dissemination, provides a unique advance in the comprehension of tumors progression and dissemination confirming the presence of CTC and its association with high risk of relapse. This functionalized surface identifying and quantifying high 5-methylcytosine CTC-subset content into the patient's blood lead significant progress in cancer risk assessment, also providing a novel therapeutic strategy.

Probing droplets on superhydrophobic surfaces by synchrotron radiation scattering techniques.

Droplets on artificially structured superhydrophobic surfaces represent quasi contact-free sample environments which can be probed by X-ray microbeams and nanobeams in the absence of obstructing walls. This review will discuss basic surface wettability concepts and introduce the technology of structuring surfaces. Quasi contact-free droplets are compared with contact-free droplets; processes related to deposition and evaporation on solid surfaces are discussed. Droplet coalescence based on the electrowetting effect allows the probing of short-time mixing and reaction processes. The review will show for several materials of biological interest that structural processes related to conformational changes, nucleation and assembly during droplet evaporation can be spatially and temporally resolved by raster-scan diffraction techniques. Orientational ordering of anisotropic materials deposited during solidification at pinning sites facilitates the interpretation of structural data.

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.

Electroless deposition and nanolithography can control the formation of materials at the nano-scale for plasmonic applications.

The new revolution in materials science is being driven by our ability to manipulate matter at the molecular level to create structures with novel functions and properties. The aim of this paper is to explore new strategies to obtain plasmonic metal nanostructures through the combination of a top down method, that is electron beam lithography, and a bottom up technique, that is the chemical electroless deposition. This technique allows a tight control over the shape and size of bi- and three-dimensional metal patterns at the nano scale. The resulting nanostructures can be used as constituents of Surface Enhanced Raman Spectroscopy (SERS) substrates, where the electromagnetic field is strongly amplified. Our results indicate that, in electroless growth, high quality metal nanostructures with sizes below 50 nm may be easily obtained. These findings were explained within the framework of a diffusion limited aggregation (DLA) model, that is a simulation model that makes it possible to decipher, at an atomic level, the rules governing the evolution of the growth front; moreover, we give a description of the physical mechanisms of growth at a basic level. In the discussion, we show how these findings can be utilized to fabricate dimers of silver nanospheres where the size and shape of those spheres is controlled with extreme precision and can be used for very large area SERS substrates and nano-optics, for single molecule detection.

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.

Nanostructured superhydrophobic substrates trigger the development of 3D neuronal networks.

The generation of 3D networks of primary neurons is a big challenge in neuroscience. Here, a novel method is presented for a 3D neuronal culture on superhydrophobic (SH) substrates. How nano-patterned SH devices stimulate neurons to build 3D networks is investigated. Scanning electron microscopy and confocal imaging show that soon after plating neurites adhere to the nanopatterned pillar sidewalls and they are subsequently pulled between pillars in a suspended position. These neurons display an enhanced survival rate compared to standard cultures and develop mature networks with physiological excitability. These findings underline the importance of using nanostructured SH surfaces for directing 3D neuronal growth, as well as for the design of biomaterials for neuronal regeneration.

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.

Microfluidic biofunctionalisation protocols to form multi-valent interactions for cell rolling and phenotype modification investigations.

In this study, we propose a fast, simple method to biofunctionalise microfluidic systems for cellomic investigations based on micro-fluidic protocols. Many available processes either require expensive and time-consuming protocols or are incompatible with the fabrication of microfluidic systems. Our method differs from the existing since it is applicable to an assembled system, uses few microlitres of reagents and it is based on the use of microbeads. The microbeads have specific surface moieties to link the biomolecules and couple cell receptors. Furthermore, the microbeads serve as arm spacer and offer the benefit of the multi-valent interaction. Microfluidics was adapted together with topology and biochemistry surface modifications to offer the microenvironment for cellomic studies. Based on this principle, we exploit the streptavidin-biotin interaction to couple antibodies to the biofunctionalised microfluidic environment within 5 h using 200 µL of reagents and biomolecules. We selected the antibodies able to form complexes with the MHC class I (MHC-I) molecules present on the cell membrane and involved in the immune surveillance. To test the microfluidic system, tumour cell lines (RMA) were rolled across the coupled antibodies to recognise and strip MHC-I molecules. As result, we show that cell rolling performed inside a microfluidic chamber functionalised with beads and the opportune antibody facilitate the removal of MHC class I molecules. We showed that the level of median fluorescent intensity of the MHC-I molecules is 300 for cells treated in a not biofunctionalised surface. It decreased to 275 for cells treated in a flat biofunctionalised surface and to 250 for cells treated on a surface where biofunctionalised microbeads were immobilised. The cells with reduced expression of MHC-I molecules showed, after cytotoxicity tests, susceptibility 3.5 times higher than normal cells.

Raman database of amino acids solutions: a critical study of extended multiplicative signal correction.

The Raman spectra of biological materials always exhibit complex profiles, constituting several peaks and/or bands which arise due to the large variety of biomolecules. The extraction of quantitative information from these spectra is not a trivial task. While qualitative information can be retrieved from the changes in peaks frequencies or from the appearance/disappearance of some peaks, quantitative analysis requires an examination of peak intensities. Unfortunately in biological samples it is not easy to identify a reference peak for normalizing intensities, and this makes it very difficult to study the peak intensities. In the last decades a more refined mathematical tool, the extended multiplicative signal correction (EMSC), has been proposed for treating infrared spectra, which is also capable of providing quantitative information. From the mathematical and physical point of view, EMSC can also be applied to Raman spectra, as recently proposed. In this work the reliability of the EMSC procedure is tested by application to a well defined biological system: the 20 standard amino acids and their combination in peptides. The first step is the collection of a Raman database of these 20 amino acids, and subsequently EMSC processing is applied to retrieve quantitative information from amino acids mixtures and peptides. A critical review of the results is presented, showing that EMSC has to be carefully handled for complex biological systems.