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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).

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.

In this study, transmission electron microscopy atomic force microscopy, and surface enhanced Raman spectroscopy are combined through a direct imaging approach, to gather structural and chemical information of complex molecular systems such as ion channels in their original plasma membrane. Customized microfabricated sample holder allows to characterize Na v channels embedded in the original plasma membrane extracted from neuronal cells that are derived from healthy human induced pluripotent stem cells. The identification of the channels is accomplished by using two different approaches, one of them widely used in cryo-EM (the particle analysis method) and the other based on a novel Zernike Polynomial expansion of the images bitmap. This approach allows to carry out a whole series of investigations, one complementary to the other, on the same sample, preserving its state as close as possible to the original membrane configuration., (© 2022 The Authors. Small Methods published by Wiley-VCH GmbH.)

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., (© 2022. The Author(s).)

In designing a new drug, considering the preferred route of administration, various requirements must be fulfilled. Active molecules pharmacokinetics should be reliable with a valuable drug profile as well as well-tolerated. Over the past 20 years, nanotechnologies have provided alternative and complementary solutions to those of an exclusively pharmaceutical chemical nature since scientists and clinicians invested in the optimization of materials and methods capable of regulating effective drug delivery at the nanometer scale. Among the many drug delivery carriers, lipid nano vesicular ones successfully support clinical candidates approaching such problems as insolubility, biodegradation, and difficulty in overcoming the skin and biological barriers such as the blood-brain one. In this review, the authors discussed the structure, the biochemical composition, and the drug delivery applications of lipid nanovesicular carriers, namely, niosomes, proniosomes, ethosomes, transferosomes, pharmacosomes, ufasomes, phytosomes, catanionic vesicles, and extracellular vesicles.

Superhydrophobic surfaces display an extraordinary repulsion to water and water-based solutions. This effect emerges from the interplay of intrinsic hydrophobicity of the surface and its morphology. These surfaces have been established for a long time and have been studied for decades. The increasing interest in recent years has been focused towards applications in many different fields and, in particular, biomedical applications. In this paper, we review the progress achieved in the last years in the fabrication of regularly patterned superhydrophobic surfaces in many different materials and their exploitation for the manipulation and characterization of biomaterial, with particular emphasis on the issues affecting the yields of the fabrication processes and the quality of the manufactured devices.

This review looks at the different approaches, techniques, and materials devoted to DNA studies. In the past few decades, DNA nanotechnology, micro-fabrication, imaging, and spectroscopies have been tailored and combined for a broad range of medical-oriented applications. The continuous advancements in miniaturization of the devices, as well as the continuous need to study biological material structures and interactions, down to single molecules, have increase the interdisciplinarity of emerging technologies. In the following paragraphs, we will focus on recent sensing approaches, with a particular effort attributed to cutting-edge techniques for structural and mechanical studies of nucleic acids.

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.

MXene-based hydrogels, a flourishing family of soft materials, have recently emerged as promising candidates for stretchable electronics. Despite recent progress, most works use MXenes as conductive nanofillers. Herein, by tuning the molecular interactions between MXene nanosheets and other constituents within the hydrogels, we demonstrate Ti 3 C 3 T x MXene can act as a versatile cross-linker to activate the fast gelation of a wide range of hydrogels, starting from various monomer- and polymer-based precursors. The gelation behavior varies significantly across hydrogels. In general, the fast gelation mechanism is attributed to the easier generation of free radicals with the help of Ti 3 C 2 T x MXene and the presence of multiscale molecular interactions between MXene and polymers. The use of MXene as a dynamic cross-linker leads to superior mechanical properties, adhesion, and self-healing ability. Owing to the inherent photothermal behavior of Ti 3 C 3 T x and the heterogeneous phase-transforming features of polymers, a polymer-MXene hydrogel is demonstrated to exhibit distinctive thermosensation-based actuation upon near-infrared illumination, accompanied by rapid shape transformation.

Mesoporous materials are structures characterized by a well-ordered large pore system with uniform porous dimensions ranging between 2 and 50 nm. Typical samples are zeolite, carbon molecular sieves, porous metal oxides, organic and inorganic porous hybrid and pillared materials, silica clathrate and clathrate hydrates compounds. Improvement in biochemistry and materials science led to the design and implementation of different types of porous materials ranging from rigid to soft two-dimensional (2D) and three-dimensional (3D) skeletons. The present review focuses on the use of three-dimensional printed (3DP) mesoporous scaffolds suitable for a wide range of drug delivery applications, due to their intrinsic high surface area and high pore volume. In the first part, the importance of the porosity of materials employed for drug delivery application was discussed focusing on mesoporous materials. At the end of the introduction, hard and soft templating synthesis for the realization of ordered 2D/3D mesostructured porous materials were described. In the second part, 3DP fabrication techniques, including fused deposition modelling, material jetting as inkjet printing, electron beam melting, selective laser sintering, stereolithography and digital light processing, electrospinning, and two-photon polymerization were described. In the last section, through recent bibliographic research, a wide number of 3D printed mesoporous materials, for in vitro and in vivo drug delivery applications, most of which relate to bone cells and tissues, were presented and summarized in a table in which all the technical and bibliographical details were reported. This review highlights, to a very cross-sectional audience, how the interdisciplinarity of certain branches of knowledge, as those of materials science and nano-microfabrication are, represent a growing valuable aid in the advanced forum for the science and technology of pharmaceutics and biopharmaceutics.

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.

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.

Polycaprolactone (PCL) is a biocompatible and biodegradable polymer widely used for the realization of 3D scaffold for tissue engineering applications. The hot embossing technique (HE) allows the obtainment of PCL scaffolds with a regular array of micro pillars on their surface. The main drawback affecting this kind of micro fabrication process is that such structural superficial details can be damaged when detaching the replica from the mold. Therefore, the present study has focused on the optimization of the HE processes through the development of an analytical model for the prediction of the demolding force as a function of temperature. This model allowed calculating the minimum demolding force to obtain regular micropillars without defects. We demonstrated that the results obtained by the analytical model agree with the experimental data. To address the importance of controlling accurately the fabricated microstructures, we seeded on the PCL scaffolds human stromal cell line (HS-5) and monocytic leukemia cell line (THP-1) to evaluate how the presence of regular or deformed pillars affect cells viability. In vitro viability results, scanning electron and fluorescence microscope imaging analysis show that the HS-5 preferentially grows on regular microstructured surfaces, while the THP-1 on irregular microstructured ones., (Copyright © 2020 Limongi, Dattola, Botta, Coluccio, Candeloro, Cucè, Scopacasa, Gallo Cantafio, Critello, Pullano, Fiorillo, Tagliaferri, Tassone, Lamanna, Di Fabrizio and Perozziello.)

Brain ischemia represents a leading cause of death and disability in industrialized countries. To date, therapeutic intervention is largely unsatisfactory and novel strategies are required for getting better protection of neurons injured by cerebral blood flow restriction. Recent evidence suggests that brain insulin leads to protection of neuronal population undergoing apoptotic cell death via modulation of oxidative stress and mitochondrial cytochrome c (CytC), an effect to be better clarified. In this work, we investigate on the effect of insulin given intracerebroventricular (ICV) before inducing a transient global ischemia by bilateral occlusion of the common carotid arteries (BCCO) in Mongolian gerbils (MG). The transient (3 min) global ischemia in MG is observed to produce neurodegenerative effect mainly into CA3 hippocampal region, 72 h after cerebral blood restriction. Intracerebroventricular microinfusion of insulin significantly prevents the apoptosis of CA3 hippocampal neurons. Histological observation, after hematoxylin and eosin staining, puts in evidence the neuroprotective role of insulin, but Raman microimaging provides a clearer insight in the CytC mechanism underlying the apoptotic process. Above all, CytC has been revealed to be an outstanding, innate Raman marker for monitoring the cells status, thanks to its resonant scattering at 530 nm of incident wavelength and to its crucial role in the early stages of cells apoptosis. These data support the hypothesis of an insulin-dependent neuroprotection and antiapoptotic mechanism occurring in the brain of MG undergoing transient brain ischemia. The observed effects occurred without any peripheral change on serum glucose levels, suggesting an alternative mechanism of insulin-induced neuroprotection.

Lipid Droplets (LDs) are emerging as crucial players in colon cancer development and maintenance. Their expression has been associated with high tumorigenicity in Cancer Stem Cells (CSCs), so that they have been proposed as a new functional marker in Colorectal Cancer Stem Cells (CR-CSCs). They are also indirectly involved in the modulation of the tumor microenvironment through the production of pro-inflammatory molecules. There is growing evidence that a possible connection between metabolic alterations and malignant transformation exists, although the effects of nutrients, primarily glucose, on the CSC behavior are still mostly unexplored. Glucose is an essential fuel for cancer cells, and the connections with LDs in the healthy and CSC populations merit to be more deeply investigated. Here, we showed that a high glucose concentration activated the PI3K/AKT pathway and increased the expression of CD133 and CD44v6 CSC markers. Additionally, glucose was responsible for the increased amount of Reactive Oxygen Species (ROS) and LDs in both healthy and CR-CSC samples. We also investigated the gene modulations following the HG treatment and found out that the healthy cell gene profile was the most affected. Lastly, Atorvastatin, a lipid-lowering drug, induced the highest mortality on CR-CSCs without affecting the healthy counterpart., Competing Interests: Authors declare no conflict of interest., (© 2019 Chongqing Medical University. Production and hosting by Elsevier B.V.)

This work presents a disposable passive microfluidic system, allowing chemotaxis studies, through the generation of a concentration gradient. The device can handle liquid flows without an external supply of pressure or electric gradients, but simply using gravity force. It is able to ensure flow rates of 10 µL/h decreasing linearly with 2.5% in 24 h. The device is made of poly(methylmethacrylate) (PMMA), a biocompatible material, and it is fabricated by micro-milling and solvent assisted bonding. It is assembled into a mini incubator, designed properly for cell biology studies in passive microfluidic devices, which provides control of temperature and humidity levels, a contamination-free environment for cells with air and 5% of CO 2 . Furthermore, the mini incubator can be mounted on standard inverted optical microscopes. By using our microfluidic device integrated into the mini incubator, we are able to evaluate and follow in real-time the migration of any cell line to a chemotactic agent. The device is validated by showing cell migration at a rate of 0.36 µm/min, comparable with the rates present in scientific literature.

The effect of direct or indirect binding of intercalant molecules on DNA structure is of fundamental importance in understanding the biological functioning of DNA. Here we report on self-suspended DNA nanobundles as ultrasensitive nanomechanical resonators for structural studies of DNA-ligand complexes. Such vibrating nanostructures represent the smallest mechanical resonator entirely composed of DNA. A correlative analysis between the mechanical and structural properties is exploited to study the intrinsic changes of double strand DNA, when interacting with different intercalant molecules (YOYO-1 and GelRed) and a chemotherapeutic drug (Cisplatin), at different concentrations. Possible implications of our findings are related to the study of interaction mechanism of a wide category of molecules with DNA, and to further applications in medicine, such as optimal titration of chemotherapeutic drugs and environmental studies for the detection of heavy metals in human serum.

A long-standing goal of neuroscience is a theory that explains the formation of the minicolumns in the cerebral cortex. Minicolumns are the elementary computational units of the mature neocortex. Here, we use zinc oxide nanowires with controlled topography as substrates for neural-cell growth. We observe that neuronal cells form networks where the networks characteristics exhibit a high sensitivity to the topography of the nanowires. For certain values of nanowires density and fractal dimension, neuronal networks express small world attributes, with enhanced information flows. We observe that neurons in these networks congregate in superclusters of approximately 200 neurons. We demonstrate that this number is not coincidental: the maximum number of cells in a supercluster is limited by the competition between the binding energy between cells, adhesion to the substrate, and the kinetic energy of the system. Since cortical minicolumns have similar size, similar anatomical and topological characteristics of neuronal superclusters on nanowires surfaces, we conjecture that the formation of cortical minicolumns is likewise guided by the interplay between energy minimization, information optimization and topology. For the first time, we provide a clear account of the mechanisms of formation of the minicolumns in the brain.

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.

The aim of this study was the design of a 3D scaffold composed of poly(vinyl) alcohol (PVA) for cardiac tissue engineering (CTE) applications. The PVA scaffold was fabricated using a combination of gas foaming and freeze-drying processes that did not need any cross-linking agents. We obtained a biocompatible porous matrix with excellent mechanical properties. We measured the stress-strain curves of the PVA scaffolds and we showed that the elastic behavior is similar to that of the extracellular matrix of muscles. The SEM observations revealed that the scaffolds possess micro pores having diameters ranging from 10 μm to 370 μm that fit to the dimensions of the cells. A further purpose of this study was to test scaffolds ability to support human induced pluripotent stem cells growth and differentiation into cardiomyocytes. As the proliferation tests show, the number of live stem cells on the scaffold after 12 days was increased with respect to the initial number of cells, revealing the cytocompatibility of the substrate. In addition, the differentiated cells on the PVA scaffolds expressed anti-troponin T, a marker specific of the cardiac sarcomere. We demonstrated the ability of the cardiomyocytes to pulse within the scaffolds. In conclusion, the developed scaffold show the potential to be used as a biomaterial for CTE applications., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Biopolymers are increasingly employed for neuroscience applications as scaffolds to drive and promote neural regrowth, thanks to their ability to mediate the upload and subsequent release of active molecules and drugs. Synthetic degradable polymers are characterized by different responses ranging from tunable distension or shrinkage to total dissolution, depending on the function they are designed for. In this paper we present a biocompatible microfabricated poly-ε-caprolactone (PCL) scaffold for primary neuron growth and maturation that has been optimized for the in vitro controlled release of brain-derived neurotrophic factor (BDNF). We demonstrate that the designed morphology confers to these devices an enhanced drug delivery capability with respect to monolithic unstructured supports. After incubation with BDNF, micropillared PCL devices progressively release the neurotrophin over 21 days in vitro. Moreover, the bioactivity of released BDNF is confirmed using primary neuronal cultures, where it mediates a consistent activation of BDNF signaling cascades, increased synaptic density, and neuronal survival. These results provide the proof-of-principle on the fabrication process of micropatterned PCL devices, which represent a promising therapeutic option to enhance neuronal regeneration after lesion and for neural tissue engineering and prosthetics.

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.

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.

Background: Human pluripotent stem cells, including embryonic stem cells and induced pluripotent stem cells, hold enormous promise for many biomedical applications, such as regenerative medicine, drug testing, and disease modeling. Although induced pluripotent stem cells resemble embryonic stem cells both morphologically and functionally, the extent to which these cell lines are truly equivalent, from a molecular point of view, remains controversial., Methods: Principal component analysis and K-means cluster analysis of collected Raman spectroscopy data were used for a comparative study of the biochemical fingerprint of human induced pluripotent stem cells and human embryonic stem cells. The Raman spectra analysis results were further validated by conventional biological assays., Results: Raman spectra analysis revealed that the major difference between human embryonic stem cells and induced pluripotent stem cells is due to the nucleic acid content, as shown by the strong positive peaks at 785, 1098, 1334, 1371, 1484, and 1575 cm -1 , which is enriched in human induced pluripotent stem cells., Conclusions: Here, we report a nonbiological approach to discriminate human induced pluripotent stem cells from their native embryonic stem cell counterparts.

We present a theoretical approach to narrow the plasmon linewidth and enhance the near-field intensity at a plasmonic dimer gap (hot spot) through coupling the electric localized surface plasmon (LSP) resonance of a silver hemispherical dimer with the resonant modes of a Fabry-Perot (FP) cavity. The strong coupling is demonstrated by the large anticrossing in the reflection spectra and a Rabi splitting of 76 meV. Up to 2-fold enhancement increase can be achieved compared to that without using the cavity. Such high field enhancement has potential applications in optics, including sensors and high resolution imaging devices. In addition, the resonance splitting allows for greater flexibility in using the same array at different wavelengths. We then further propose a practical design to realize such a device and include dimers of different shapes and materials.

Nanotechnology allows the realization of new materials and devices with basic structural unit in the range of 1-100 nm and characterized by gaining control at the atomic, molecular, and supramolecular level. Reducing the dimensions of a material into the nanoscale range usually results in the change of its physiochemical properties such as reactivity, crystallinity, and solubility. This review treats the convergence of last research news at the interface of nanostructured biomaterials and tissue engineering for emerging biomedical technologies such as scaffolding and tissue regeneration. The present review is organized into three main sections. The introduction concerns an overview of the increasing utility of nanostructured materials in the field of tissue engineering. It elucidates how nanotechnology, by working in the submicron length scale, assures the realization of a biocompatible interface that is able to reproduce the physiological cell-matrix interaction. The second, more technical section, concerns the design and fabrication of biocompatible surface characterized by micro- and submicroscale features, using microfabrication, nanolithography, and miscellaneous nanolithographic techniques. In the last part, we review the ongoing tissue engineering application of nanostructured materials and scaffolds in different fields such as neurology, cardiology, orthopedics, and skin tissue regeneration.

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.

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).

Pedot: PSS is a conductive polymer that can be integrated into last generation Organic Electrochemical Transistor (OECT) devices for biological inspection, identification and analysis. While a variety of reports in literature demonstrated the chemical and biological sensitivity of these devices, still their ability in resolving complex mixtures remains controversial. Similar OECT devices display good time dynamics behavior but lack spatial resolution. In this work, we integrated, Pedot: PSS with patterns of super-hydrophobic pillars in which a finite number of those pillars is independently controlled for site-selective measurement of a solution. We obtained a multifunctional, hierarchical OECT device that bridges the micro- to the nano-scales for specific, combined time and space resolved analysis of the sample. Due to super-hydrophobic surface properties, the biological species in the drop are driven by convection, diffusion, and the externally applied electric field: the balance/unbalance between these forces will cause the molecules to be transported differently within its volume depending on particle size thus realizing a size-selective separation. Within this framework, the separation and identification of two different molecules, namely Cetyl Trimethyl Ammonium Bromid (CTAB) and adrenaline, in a biological mixture have been demonstrated, showing that geometrical control at the micro-nano scale impart unprecedented selectivity to the devices.

Neurons are specialized, electrically excitable cells which use electrical to chemical signals to transmit and elaborate information. Understanding how the cooperation of a great many of neurons in a grid may modify and perhaps improve the information quality, in contrast to few neurons in isolation, is critical for the rational design of cell-materials interfaces for applications in regenerative medicine, tissue engineering, and personalized lab-on-a-chips. In the present paper, we couple an integrate-and-fire model with information theory variables to analyse the extent of information in a network of nerve cells. We provide an estimate of the information in the network in bits as a function of cell density and short-term depression time. In the model, neurons are connected through a Delaunay triangulation of not-intersecting edges; in doing so, the number of connecting synapses per neuron is approximately constant to reproduce the early time of network development in planar neural cell cultures. In simulations where the number of nodes is varied, we observe an optimal value of cell density for which information in the grid is maximized. In simulations in which the posttransmission latency time is varied, we observe that information increases as the latency time decreases and, for specific configurations of the grid, it is largely enhanced in a resonance effect.

The redox state of the cell is involved in the regulation of many physiological functions as well as in the pathogenesis of several diseases, and is strictly dependent on the amount of iron in its catalytically active state. Alterations of iron homeostasis determine increased steady-state concentrations of Reactive Oxygen Species (ROS) that cause lipid peroxidation, DNA damage and altered protein folding. Ferritin keeps the intracellular iron in a non-toxic and readily available form and consequently plays a central role in iron and redox homeostasis. The protein is composed by 24 subunits of the H- and L-type, coded by two different genes, with structural and functional differences. The aim of this study was to shed light on the role of the single H ferritin subunit (FHC) in keeping the native correct protein three-dimensional structure. To this, we performed Raman spectroscopy on protein extracts from K562 cells subjected to FHC silencing. The results show a significant increase in the percentage of disordered structures content at a level comparable to that induced by H2O2 treatment in control cells. ROS inhibitor and iron chelator were able to revert protein misfolding. This integrated approach, involving Raman spectroscopy and targeted-gene silencing, indicates that an imbalance of the heavy-to-light chain ratio in the ferritin composition is able to induce severe but still reversible modifications in protein folding and uncovers new potential pathogenetic mechanisms associated to intracellular iron perturbation., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Advanced optical materials or interfaces are gaining attention for diagnostic applications. However, the achievement of large device interface as well as facile surface functionalization largely impairs their wide use. The present work is aimed to address different innovative aspects related to the fabrication of large-area 3D plasmonic arrays, their direct and easy functionalization with capture elements, and their spectroscopic verifications through enhanced Raman and enhanced fluorescence techniques. In detail, we have investigated the effect of a Au-based nanoCone array, fabricated by means of direct nanoimprint technique over large area (mm(2)), on protein capturing and on the enhancement in optical signal. A selective functionalization of gold surfaces was proposed by using a peptide (AuPi3) previously selected by phage display. In this regard, two different sequences, labeled with fluorescein and biotin, were chemisorbed on metallic surfaces. The presence of Au nanoCones array consents an enhancement in electric field on the apex of cone, enabling the detection of molecules. We have witnessed around 12-fold increase in fluorescence intensity and SERS enhancement factor around 1.75 × 10(5) with respect to the flat gold surface. Furthermore, a sharp decrease in fluorescence lifetime over nanoCones confirms the increase in radiative emission (i.e., an increase in photonics density at the apex of cones).

Control of the architecture and electromagnetic behavior of nanostructures offers the possibility of designing and fabricating sensors that, owing to their intrinsic behavior, provide solutions to new problems in various fields. We show detection of peptides in multicomponent mixtures derived from human samples for early diagnosis of breast cancer. The architecture of sensors is based on a matrix array where pixels constitute a plasmonic device showing a strong electric field enhancement localized in an area of a few square nanometers. The method allows detection of single point mutations in peptides composing the BRCA1 protein. The sensitivity demonstrated falls in the picomolar (10(-12) M) range. The success of this approach is a result of accurate design and fabrication control. The residual roughness introduced by fabrication was taken into account in optical modeling and was a further contributing factor in plasmon localization, increasing the sensitivity and selectivity of the sensors. This methodology developed for breast cancer detection can be considered a general strategy that is applicable to various pathologies and other chemical analytical cases where complex mixtures have to be resolved in their constitutive components.

The structure of DNA was determined in 1953 by x-ray fiber diffraction. Several attempts have been made to obtain a direct image of DNA with alternative techniques. The direct image is intended to allow a quantitative evaluation of all relevant characteristic lengths present in a molecule. A direct image of DNA, which is different from diffraction in the reciprocal space, is difficult to obtain for two main reasons: the intrinsic very low contrast of the elements that form the molecule and the difficulty of preparing the sample while preserving its pristine shape and size. We show that through a preparation procedure compatible with the DNA physiological conditions, a direct image of a single suspended DNA molecule can be obtained. In the image, all relevant lengths of A-form DNA are measurable. A high-resolution transmission electron microscope that operates at 80 keV with an ultimate resolution of 1.5 Å was used for this experiment. Direct imaging of a single molecule can be used as a method to address biological problems that require knowledge at the single-molecule level, given that the average information obtained by x-ray diffraction of crystals or fibers is not sufficient for detailed structure determination, or when crystals cannot be obtained from biological molecules or are not sufficient in understanding multiple protein configurations.

We have shown earlier that overexpression of the human mitochondrial ribosomal protein MRPS18-2 (S18-2) led to immortalization of primary rat embryonic fibroblasts. The derived cells expressed the embryonic stem cell markers, and cellular pathways that control cell proliferation, oxidative phosphorylation, cellular respiration, and other redox reactions were activated in the immortalized cells.Here we report that, upon overexpression of S18-2 protein, primary rat skin fibroblasts underwent cell transformation. Cells passed more than 300 population doublings, and two out of three tested clones gave rise to tumors in experimental animals. Transformed cells showed anchorage-independent growth and loss of contact inhibition; they expressed epithelial markers, such as E-cadherin and β-catenin. Transformed cells showed increased telomerase activity, disturbance of the cell cycle, and chromosomal instability. Taken together, our data suggest that S18-2 is a newly identified oncoprotein that may be involved in cancerogenesis.

The evaporation of single droplets of colloidal tobacco mosaic virus (TMV) nanoparticles on a superhydrophobic surface with a hexagonal pillar-pattern results in the formation of coffee-ring type residues. We imaged surface features by optical, scanning electron, and atomic force microscopies. Bulk features were probed by raster-scan X-ray nanodiffraction. At ∼100 pg/μL nanoparticle concentration, the rim of the residue connects to neighboring pillars via fibrous extensions containing flow-aligned crystalline domains. At ∼1 pg/μL nanoparticle concentration, nanofilaments of ≥80 nm diameter and ∼20 μm length are formed, extending normal to the residue-rim across a range of pillars. X-ray scattering is dominated by the nanofilament form-factor but some evidence for crystallinity has been obtained. The observation of sheets composed of stacks of self-assembled nanoparticles deposited on pillars suggests that the nanofilaments are drawn from a structured droplet interface.

Confocal microscopy imaging of cells allows to visualize the presence of specific antigens by using fluorescent tags or fluorescent proteins, with resolution of few hundreds of nanometers, providing their localization in a large field-of-view and the understanding of their cellular function. Conversely, in scanning electron microscopy (SEM), the surface morphology of cells is imaged down to nanometer scale using secondary electrons. Combining both imaging techniques have brought to the correlative light and electron microscopy, contributing to investigate the existing relationships between biological surface structures and functions. Furthermore, in SEM, backscattered electrons (BSE) can image local compositional differences, like those due to nanosized gold particles labeling cellular surface antigens. To perform SEM imaging of cells, they could be grown on conducting substrates, but obtaining images of limited quality. Alternatively, they could be rendered electrically conductive, coating them with a thin metal layer. However, when BSE are collected to detect gold-labeled surface antigens, heavy metals cannot be used as coating material, as they would mask the BSE signal produced by the markers. Cell surface could be then coated with a thin layer of chromium, but this results in a loss of conductivity due to the fast chromium oxidation, if the samples come in contact with air. In order to overcome these major limitations, a thin layer of indium-tin-oxide was deposited by ion-sputtering on gold-decorated HeLa cells and neurons. Indium-tin-oxide was able to provide stable electrical conductivity and preservation of the BSE signal coming from the gold-conjugated markers., (© 2015 Wiley Periodicals, Inc.)

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.

The human brain is a tightly interweaving network of neural cells where the complexity of the network is given by the large number of its constituents and its architecture. The topological structure of neurons in the brain translates into its increased computational capabilities, low energy consumption, and nondeterministic functions, which differentiate human behavior from artificial computational schemes. In this manuscript, we fabricated porous silicon chips with a small pore size ranging from 8 to 75 nm and large fractal dimensions up to Df ∼ 2.8. In culturing neuroblastoma N2A cells on the described substrates, we found that those cells adhere more firmly to and proliferate on the porous surfaces compared to the conventional nominally flat silicon substrates, which were used as controls. More importantly, we observed that N2A cells on the porous substrates create highly clustered, small world topology patterns. We conjecture that neurons with a similar architecture may elaborate information more efficiently than in random or regular grids. Moreover, we hypothesize that systems of neurons on nano-scale geometry evolve in time to form networks in which the propagation of information is maximized.

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.

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., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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., (© 2014 The Authors. STEM CELLS Published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.)

In our body, cells are continuously exposed to physical forces that can regulate different cell functions such as cell proliferation, differentiation and death. In this work, we employed two different strategies to mechanically stress cancer cells. The cancer and healthy cell populations were treated either with mechanical stress delivered by a micropump (fabricated by deep X-ray nanolithography) or by ultrasound wave stimuli. A specific down-regulation of Major Histocompatibility Complex (MHC) class I molecules expression on cancer cell membrane compared to different kinds of healthy cells (fibroblasts, macrophages, dendritic and lymphocyte cells) was observed, stimulating the cells with forces in the range of nano-newton, and pressures between 1 and 10 bar (1 bar = 100.000 Pascal), depending on the devices used. Moreover, Raman spectroscopy analysis, after mechanical treatment, in the range between 700-1800 cm(-1), indicated a relative concentration variation of MHC class I. PCA analysis was also performed to distinguish control and stressed cells within different cell lines. These mechanical induced phenotypic changes increase the tumor immunogenicity, as revealed by the related increased susceptibility to Natural Killer (NK) cells cytotoxic recognition.

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., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Super-hydrophobic surfaces are bio-inspired interfaces with a superficial texture that, in its most common evolution, is formed by a periodic lattice of silicon micro-pillars. Similar surfaces reveal superior properties compared to conventional flat surfaces, including very low friction coefficients. In this work, we modified meso-porous silicon micro-pillars to incorporate networks of metal nano-particles into the porous matrix. In doing so, we obtained a multifunctional-hierarchical system in which (i) at a larger micrometric scale, the super-hydrophobic pillars bring the molecules dissolved in an ultralow-concentration droplet to the active sites of the device, (ii) at an intermediate meso-scale, the meso-porous silicon film adsorbs the low molecular weight content of the solution and, (iii) at a smaller nanometric scale, the aggregates of silver nano-particles would measure the target molecules with unprecedented sensitivity. In the results, we demonstrated how this scheme can be utilized to isolate and detect small molecules in a diluted solution in very low abundance ranges. The presented platform, coupled to Raman or other spectroscopy techniques, is a realistic candidate for the protein expression profiling of biological fluids.

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.

Plasmonic nanostar-dimers, decoupled from the substrate, have been fabricated by combining electron-beam lithography and reactive-ion etching techniques. The 3D architecture, the sharp tips of the nanostars and the sub-10 nm gap size promote the formation of giant electric-field in highly localized hot-spots. The single/few molecule detection capability of the 3D nanostar-dimers has been demonstrated by Surface-Enhanced Raman Scattering., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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.