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Amino-terminated surfaces can be effectively obtained by means of silanizing agents, realizing surfaces suitable for the purification of biomarkers of several pathologies. Since the level of biomarkers, such as microRNAs and cell-free DNA, into circulation may be extremely low, new and ameliorated capturing molecules and protocols are highly required. In this work, a new silane, acetone-imine propyl trimethoxysilane (AIPTMS), is synthesized with a simple and elegant reaction, via the nucleophilic addition of the primary amino group to the carbonyl group of acetone. AIPTMS and APTMS were used to silanize silicon oxide surfaces, which were characterized chemically (XPS) and morphologically (AFM). The two types of surfaces were chemically similar, but behaved very differently both for surface morphology and functional properties. The AIPTMS-modified surface was indeed very smooth and homogeneous with respect to the APTMS-modified surface. Moreover, the AIPTMS surface captured larger amounts of nucleic acids almost immediately after preparation, while APTMS-based functional surfaces needed longer time to reach comparable efficiency. AIPTMS shows several advantages over standard aminosilanes, as it realizes a more homogeneous surface coverage that, in turn, produces an improved response towards the capture of nucleic acids. AIPTMS is a very promising reagent for the reliable and reproducible preparation of active biofunctional surfaces for the purification and analysis of circulating biomarkers.

Polydimethylsiloxane (PDMS) is a well-known biocompatible polymer employed for many applications in the biomedical field. In this study, the biocompatibility and versatility of PDMS was tested setting up a microdevice devoted to the purification and analysis of nucleic acids. The PDMS microdevice was demonstrated to successfully fulfill all requirements of genetic analyses such as genotyping and pathogen DNA identification both in multiplex and real-time PCR, suggesting the possibility to carry out a molecular test directly on-chip. Moreover, the PDMS microdevice was successfully applied to the purification and detection of disease biomarkers, such as microRNAs related to cancer or heart disease. On-chip microRNA purification was demonstrated starting from clinically relevant samples, i.e. plasma, serum, tissue biopsies. Significantly, the purification and the transcription of microRNA into cDNA occur in the same PDMS chamber, saving time and labor for the overall analysis. Again, the PDMS microdevice was confirmed as a notable candidate for compact, rapid, easy-to-use molecular tests.

A specific aptameric sequence has been immobilized on short polyethyleneglycol (PEG) interface on gold nano-film deposited on a D-shaped plastic optical fiber (POFs) probe, and the protein binding has been monitored exploiting the very sensitive surface plasmon resonance (SPR) phenomenon. The receptor-binding domain (RBD) of the SARS-CoV-2 spike glycoprotein has been specifically used to develop an aptasensor. Surface analysis techniques coupled to fluorescence microscopy and plasmonic analysis have been utilized to characterize the biointerface. Spanning a wide protein range (25 ÷ 1000 nM), the SARS-Cov-2 spike protein was detected with a Limit of Detection (LoD) of about 37 nM. Different interferents (BSA, AH1N1 hemagglutinin protein and MERS spike protein) have been tested confirming the specificity of our aptasensor. Finally, a preliminary test in diluted human serum encouraged its application in a point-of-care device, since POF-based aptasensor represent a potentially low-cost compact biosensor, characterized by a rapid response, a small size and could be an ideal laboratory portable diagnostic tool., Graphical abstract Image 1

The neuroscience field has increased enormously over the last decades, achieving the possible real application of neuronal cultures not only for reproducing neural architectures resembling in vivo tissues, but also for the development of functional devices. In this context, surface patterning for cell confinement is crucial, and new active materials together with new protocols for preparing substrates suitable for confining cells, guiding their processes in the desired configuration are extremely appreciated. Here, TiO2 sol-gel derived films were selected as proof-of-concept materials to grow neurons in suitable confined configurations, taking advantage of the biocompatible properties of modified TiO2 substrates. TiO2 sol-gel derived films were made compatible with the growth of neurons thanks to a stable and controlled poly-lysine coating, obtained by silanization chemistry and streptavidin-biotin interactions. Moreover, a spotting protocol, here described and optimized, allowed the simple preparation of arrays of neurons, where cell adhesion was guided in specific areas and the neurites development driven in the desired arrangement. The resulting arrays were successfully tested for the growth and differentiation of neurons, demonstrating the possible adhesion of cells in specific areas of the film, therefore paving the way to applications such as the direct growth of excitable cells nearby electrodes of devices, with an evident enhancement of cell-electrodes communication.

We report on chemical, biochemical and physical sensors based on whispering gallery mode resonators. We characterized the resonators, improved the mitigation of fluctuations, and developed different types of optical sensors, labelled and label-free sensors for chemical and biochemical sensing, and pressure, temperature and nanoparticle sensing., Franco Cosi is gratefully acknowledged for his help in the device fabrication. Franco Quercioli and Pablo Loza-Alvarez are gratefully acknowledged for allowing us to use their nonlinear microscopes.

Extracellular vesicles (EVs) are membranous structures that cells massively release in extracellular fluids. EVs are cargo of cellular components such as lipids, proteins, and nucleic acids that can work as a formidable source in liquid biopsy studies searching for disease biomarkers. We recently demonstrated that nickel-based isolation (NBI) is a valuable method for fast, efficient, and easy recovery of heterogeneous EVs from biological fluids. NBI exploits nickel cations to capture negatively charged vesicles. Then, a mix of balanced chelating agents elutes EVs while preserving their integrity and stability in solution. Here, we describe steps and quality controls to functionalize a matrix of agarose beads, obtain an efficient elution of EVs, and extract nucleic acids carried by them. We demonstrate the versatility of NBI method in isolating EVs from media of primary mouse astrocytes, from human blood, urine, and saliva processed in parallel, as well as outer membrane vesicles (OMVs) from cultured Gram-negative bacteria.

We report a comprehensive study of the biocompatibility and neurocompatibility of titanium dioxide films (TiO2) prepared by Pulsed Microplasma Cluster Source (PMCS). This technique uses supersonic pulsed beams seeded by clusters of the metal oxide synthesized in a plasma discharge. The final stoichiometry of the TiO2 thin films is tuned changing the gas mixture, achieving stoichiometric or oxygen overstoichiometric films. All the films showed consistent biocompatibility and a spontaneous absorption of poly-d-lysine (PDL) that favors the adhesion and growth of murine cortical neurons. Moreover, the bioelectrical activity of the neuronal culture grown on the TiO2 film can be modulated by changing the chemistry of the surface. This work paves the way to develop a bio-hybrid neuromorphic device, where viable nerve cells are grown directly over a titanium dioxide film showing a network of memristors.

RALY is a member of the heterogeneous nuclear ribonucleoprotein family (hnRNP), a large family of RNA-binding proteins involved in many aspects of RNA metabolism. Although RALY interactome has been recently characterized, a comprehensive global analysis of RALY-associated RNAs is lacking and the biological function of RALY remains elusive. Here, we performed RIP-seq analysis to identify RALY interacting RNAs and assessed the role of RALY in gene expression. We demonstrate that RALY binds specific coding and non-coding RNAs and associates with translating mRNAs of mammalian cells. Among the identified transcripts, we focused on ANXA1 and H1FX mRNAs, encoding for Annexin A1 and for the linker variant of the histone H1X, respectively. Both proteins are differentially expressed by proliferating cells and are considered as markers for tumorigenesis. We demonstrate that cells lacking RALY expression exhibit changes in the levels of H1FX and ANXA1 mRNAs and proteins in an opposite manner. We also provide evidence for a direct binding of RALY to the U-rich elements present within the 3΄UTR of both transcripts. Thus, our results identify RALY as a poly-U binding protein and as a regulator of H1FX and ANXA1 in mammalian cells.

Background Extracellular vesicles (EVs) are secreted membranous particles intensively studied for their potential cargo of diagnostic markers. Efficient and cost-effective isolation methods need to be established for the reproducible and high-throughput study of EVs in the clinical practice. Methods We designed the nickel-based isolation (NBI) to rapidly isolate EVs and combined it with newly-designed amplified luminescent proximity homogeneous assay or digital PCR to detect biomarkers of clinical utility. Findings From plasma of 46 healthy donors, we systematically recovered small EV (~250 nm of mean diameter; ~3 × 1010/ml) and large EV (~560 nm of mean diameter; ~5 × 108/ml) lineages ranging from 50 to 700 nm, which displayed hematopoietic/endothelial cell markers that were also used in spike-in experiments using EVs from tumor cell lines. In retrospective studies, we detected picomolar concentrations of prostate-specific membrane antigen (PSMA) in fractions of EVs isolated from the plasma of prostate cancer patients, discriminating them from control subjects. Directly from oil-encapsulated EVs for digital PCR, we identified somatic BRAF and KRAS mutations circulating in the plasma of metastatic colorectal cancer (CRC) patients, matching 100% of concordance with tissue diagnostics. Importantly, with higher sensitivity and specificity compared with immuno-isolated EVs, we revealed additional somatic alterations in 7% of wild-type CRC cases that were subsequently validated by further inspections in the matched tissue biopsies. Interpretation We propose NBI-combined approaches as simple, fast, and robust strategies to probe the tumor heterogeneity and contribute to the development of EV-based liquid biopsy studies. Fund Associazione Italiana per la Ricerca sul Cancro (AIRC), Fondazione Cassa di Risparmio Trento e Rovereto (CARITRO), and the Italian Ministero Istruzione, Universita e Ricerca (Miur).

In this paper, we described a versatile two steps approach for the realization of silica inverse opals functionalized with DNA-aptamers labelled with Cy3 fluorophore. The co-assembly method was successfully employed for the realization of high quality inverse silica opal, whilst the inverse network was functionalized via epoxy chemistry. Morphological and optical assessment revealed the presence of large ordered domains with a transmission band gap depth of 32%, after the functionalization procedure. Finite Difference Time-Domain (FDTD) simulations confirmed the high optical quality of the inverse opal realized. Photoluminescence measurements evidenced the effective immobilization of DNA-aptamer molecules labelled with Cy3 throughout the entire sample thickness. This assumption was verified by the inhibition of the fluorescence of Cy3 fluorophore tailoring the position of the photonic band gap of the inverse opal. The modification of the fluorescence could be justified by a variation in the density of states (DOS) calculated by the Plane Wave Expansion (PWE) method. Finally, the development of the aforementioned approach could be seen as proof of the concept experiment, suggesting that this type of system may act as a suitable platform for the realization of fluorescence-based bio-sensors.

This study explores the feasibility to use electroconductive Ti-doped polycrystalline diamond layers as scaffolds for tissue engineering. The synthesis of the diamond-based materials is accomplished in a HFCVD reactor where Ti(IV) acetyl acetonate powders are delivered by N2 fluxes to the growing diamond phase. In-depth investigations (Raman spectroscopy, SEM, AFM, XRD, XPS) allowed the characterization of the morphological/structural/compositional features and the properties of charge transport (KPFM, I-V) induced in the diamond layers by the incorporation of Ti-species. The bioactivity of the Ti-doped diamond surface was verified investigating the growth of MG-63 osteoblast-like cells by using MTT assays and confocal microscopy. The study evidenced a net increase of cell replication rate on diamond scaffolds after 4 days of incubation. After 6-days incubation, the cell growth on the Ti-doped diamond scaffolds increased up to 150% compared with the reference polystyrene tissue culture vessel, with a dominant presence of cells in active division. The cell behavior is discussed and related to the structural and functional surface properties of the Ti-diamond systems, acting as bioactive platforms able to offer an extremely beneficial environment for cell proliferation and viability. © 2020 Elsevier B.V.

The detection of low abundant biomarkers, such as circulating microRNAs, demands innovative detection methods with increased resolution, sensitivity and specificity. Here, a biofunctional surface was implemented for the selective capture of microRNAs, which were detected through fluorescence enhancement directly on a photonic crystal. To set up the optimal biofunctional surface, epoxy-coated commercially available microscope slides were spotted with specific anti-microRNA probes. The optimal concentration of probe as well as of passivating agent were selected and employed for titrating the microRNA hybridization. Cross-hybridization of different microRNAs was also tested, resulting negligible. Once optimized, the protocol was adapted to the photonic crystal surface, where fluorescent synthetic miR-16 was hybridized and imaged with a dedicated equipment. The photonic crystal consists of a dielectric multilayer patterned with a grating structure. In this way, it is possible to take advantage from both a resonant excitation of fluorophores and an angularly redirection of the emitted radiation. As a result, a significant fluorescence enhancement due to the resonant structure is collected from the patterned photonic crystal with respect to the outer non-structured surface. The dedicated read-out system is compact and based on a wide-field imaging detection, with little or no optical alignment issues, which makes this approach particularly interesting for further development such as for example in microarray-type bioassays. (C) 2016 Elsevier B.V. All rights reserved.

Many efforts have been spent in the last decade for the development of nanoscale synaptic devices integrated into neuromorphic circuits, trying to emulate the behavior of natural synapses. The study of brain properties with the standard approaches based on biocompatible electrodes coupled to conventional electronics, however, presents strong limitations, which in turn could be overcame by the in-situ growth of neuronal networks coupled to memristive devices. To meet this challenging task, here two different chips were designed and fabricated for culturing neuronal cells and sensing their electrophysiological activity. The first chip was designed to be connected to an external memristor, while the second chip was coated with TiO2 films owning memristive properties. The biocompatibility of chips was preliminary analyzed by culturing the hybrid motor-neuron cell line NSC-34 and by measuring the electrical activity of cells interfacing the chip with a standard patch-clamp setup. Next, neurons were seeded on chips and their activity measured with the same setup. For both cell types total current and voltage responses were evoked and recorded with optimal results with no breakdowns. In addition, an external stimulation was applied to cells through chip electrodes, being effective and causing no damage or pitfalls to the cells. Finally, the whole bio-hybrid system, i.e. the chip interconnected with a commercial memristor, was tested with promising results. Spontaneous electrical activity of neurons grown on the chip was indeed present and this signal was collected and sent to the memristor, changing its state. Taken together, we demonstrated the ability of memristor to work with a synaptic/plastic response together with natural systems, opening the way for the further implementation of basic computing elements able to perform both storage and processing of data, as in natural neurons.

Topology affects physical and biological properties of DNA and impacts fundamental cellular processes, such as gene expression, genome replication, chromosome structure and segregation. In all organisms DNA topology is carefully modulated and the supercoiling degree of defined genome regions may change according to physiological and environmental conditions. Elucidation of structural properties of DNA molecules with different topology may thus help to better understand genome functions. Whereas a number of structural studies have been published on highly negatively supercoiled DNA molecules, only preliminary observations of highly positively supercoiled are available, and a description of DNA structural properties over the full range of supercoiling degree is lacking. Atomic Force Microscopy (AFM) is a powerful tool to study DNA structure at single molecule level. We here report a comprehensive analysis by AFM of DNA plasmid molecules with defined supercoiling degree, covering the full spectrum of biologically relevant topologies, under different observation conditions. Our data, supported by statistical and biochemical analyses, revealed striking differences in the behavior of positive and negative plasmid molecules.

The demand of miniaturized, accurate and robust micro-tools for minimally invasive surgery or in general for micro-manipulation, has grown tremendously in recent years. To meet this need, a new-concept comb-driven microgripper was designed and fabricated. Two microgripper prototypes differing for both the number of links and the number of conjugate surface flexure hinges are presented. Their design takes advantage of an innovative concept based on the pseudo-rigid body model, while the study of microgripper mechanical potentialities in different configurations is supported by finite elements' simulations. These microgrippers, realized by the deep reactive-ion etching technology, are intended as micro-tools for tissue or cell manipulation and for minimally invasive surgery; therefore, their biocompatibility in terms of protein fouling was assessed. Serum albumin dissolved in phosphate buffer was selected to mimic the physiological environment and its adsorption on microgrippers was measured. The presented microgrippers demonstrated having great potential as biomedical tools, showing a modest propensity to adsorb proteins, independently from the protein concentration and time of incubation.

In this paper, we described a versatile two steps approach for the realization of silica inverse opals functionalized with DNA-aptamers labelled with Cy3 fluorophore. The co-assembly method was successfully employed for the realization of high quality inverse silica opal, whilst the inverse network was functionalized via epoxy chemistry. Morphological and optical assessment revealed the presence of large ordered domains with a transmission band gap depth of 32%, after the functionalization procedure. Finite Difference Time-Domain (FDTD) simulations confirmed the high optical quality of the inverse opal realized. Photoluminescence measurements evidenced the effective immobilization of DNA-aptamer molecules labelled with Cy3 throughout the entire sample thickness. This assumption was verified by the inhibition of the fluorescence of Cy3 fluorophore tailoring the position of the photonic band gap of the inverse opal. The modification of the fluorescence could be justified by a variation in the density of states (DOS) calculated by the Plane Wave Expansion (PWE) method. Finally, the development of the aforementioned approach could be seen as proof of the concept experiment, suggesting that this type of system may act as a suitable platform for the realization of fluorescence-based bio-sensors.

In stressed organisms, strategic proteins are selectively translated even if the global process of protein synthesis is compromised. The determination of protein concentrations in tissues of non-model organisms (thus with limited genomic information) is challenging due to the absence of specific antibodies. Moreover, estimating protein levels quantifying transcriptional responses may be misleading, because translational control mechanisms uncouple protein and mRNAs abundances. Translational control is increasingly recognized as a hub where regulation of gene expression converges to shape proteomes, but it is almost completely overlooked in molecular ecology studies. An interesting approach to study translation and its control mechanisms is the analysis of variations of gene-specific translational efficiencies by quantifying mRNAs associated to ribosomes. In this paper, we propose a robust and streamlined pipeline for purifying ribosome-associated mRNAs and calculating global and gene-specific translation efficiencies from non-model insect’s species. This method might found applications in molecular ecology to study responses to environmental stressors in non-model organisms.

Recurrent supra-ribosomal building blocks separated by ribosome-free mRNA regions form polysomes and reflect cellular translational activity., Translation is increasingly recognized as a central control layer of gene expression in eukaryotic cells. The overall organization of mRNA and ribosomes within polysomes, as well as the possible role of this organization in translation are poorly understood. Here we show that polysomes are primarily formed by three distinct classes of ribosome assemblies. We observe that these assemblies can be connected by naked RNA regions of the transcript. We show that the relative proportions of the three classes of ribosome assemblies reflect, and probably dictate, the level of translational activity. These results reveal the existence of recurrent supra-ribosomal building blocks forming polysomes and suggest the presence of unexplored translational controls embedded in the polysome structure.

The biocompatibility of an implant depends upon the material it is composed of, in addition to the prosthetic device's morphology, mechanical and surface properties. Properties as porosity and pore size should allow, when required, cells penetration and proliferation. Stiffness and strength, that depend on the bulk characteristics of the material, should match the mechanical requirements of the prosthetic applications. Surface properties should allow integration in the surrounding tissues by activating proper communication pathways with the surrounding cells. Bulk and surface properties are not interconnected, and for instance a bone prosthesis could possess the necessary stiffness and strength for the application omitting out prerequisite surface properties essential for the osteointegration. In this case, surface treatment is mandatory and can be accomplished using various techniques such as applying coatings to the prosthesis, ion beams, chemical grafting or modification, low temperature plasma, or a combination of the aforementioned. Low temperature plasma-based techniques have gained increasing consensus for the surface modification of biomaterials for being effective and competitive compared to other ways to introduce surface functionalities. In this paper we review plasma processing techniques and describe potentialities and applications of plasma to tailor the interface of biomaterials.

We report a comprehensive study of the biocompatibility and neurocompatibility of titanium dioxide films (TiO

A reliable clinical assay based on circulating microRNAs (miRNAs) as biomarkers is highly required. Microdevices offer an attractive solution as a fast and inexpensive way of concentrating these biomarkers from a low sample volume. A previously developed polydimethylsiloxane (PDMS) microdevice able to purify and detect circulating miRNAs was here optimized. The optimization of the morphological and chemical surface properties by nanopatterning and functionalization is presented. Surfaces were firstly characterized by atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS), fluorescamine assay and s-SDTB (sulphosuccinimidyl-4-o-(4,4-dimethoxytrityl) butyrate) assay and subsequently tested for their capacity to adsorb a fluorescent miRNA. From our analysis, modification of surface charge with 0.1% APTMS ((3-Aminopropyl)trimethoxysilane) and 0.9% PEG-s (2-[Methoxy-(polyethyleneoxy)propyl]trimethoxysilane) performed at 60 °C for 10 min was identified as the best purification condition. Our optimized microdevice integrated with real-time PCR detection, was demonstrated to selectively purify both synthetic and natural circulating miRNAs with a sensitivity of 0.01 pM.

A new communication route among cells was reported in recent years, via extracellular vesicles and their cargo. Exosomes in particular are attracting increasing interest as privileged mediators of this cell communication route. The exosome-mediated transfer of nucleic acids, especially of microRNAs, is particularly promising for their use both as biomarkers of pathologies and as a therapeutic tool. Here, a simplified model of interaction among cells, microRNAs and vesicles is studied using a biophysical approach. A synthetic and fluorescent microRNA (i.e. miR-1246 conjugated with TAMRA) was selected to model cell communication, monitoring its internalization in cells. The fluorescent miR-1246, either naked or included in synthetic or natural vesicles, was incubated with human breast adenocarcinoma cells (MCF7) for different times. A comparison between this human microRNA and its DNA copy or an exogenous microRNA (from Caenorhabditis elegans) allowed assessment of the specificity of the information transfer through microRNAs, and especially associated with exosomes. The uptake of naked miR-1246 was indeed higher both in terms of number of targeted cells and intensity of fluorescence signal with respect to the other nucleic acids tested. The same occurred with miR-1246 loaded exosomes, evidencing a specific uptake only partially due to the lipidic components and present only when the human microRNA was loaded in exosomes, which were themselves derived from the same MCF7 cells.

We report the development of sol-gel derived TiO2 thin films with adjustable and defined properties suitable for memristive cell fabrication. Memristive cells were developed by the sol-gel deposition of titania onto SiO2/Ti/Pt engineered electrodes via spin coating, followed by diverse curing and annealing procedures. The influence of the processing conditions and the sol's chemical composition on the film properties, and therefore on the memristive response, was studied by micro-Raman and transmission spectroscopies, profilometry, ellipsometry, X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), and X-ray absorption and diffraction spectroscopies (XAS and XRD). A memristive response was acquired from a number of these cells, revealing a dependence of the electrical behavior on minor changes in the TiO2 structure, electroforming parameters, and architecture. Thus, these properties provide a handle for fine-tuning electrical performance.

Economical, religious, and health reasons demand an accurate control of food in order to protect consumers from falsely labeled products. Meat in particular is easily susceptible to fraudulent labeling, mainly through contamination with species of lower value. New methods and protocols for rapid, sensitive and reliable identification of extraneous species in food are therefore required. The miniaturization and optimization of analytical methodologies are powerful tools in this direction, especially when connected to Lab-on-a-chip (LOC) microdevices. LOCs possess many advantages, such as the reduction of the analysis cost, the possibility to save time and labor, the easiness of use and not last, the possibility to bring a complex technique out of the laboratory. Here we present a new concept for the food quality control, i.e. the use of LOC for the detection of exogenous DNA in meat via on-chip PCR in real-time. LOC surfaces were treated with different coatings in order to optimize the DNA extraction directly from meat homogenates (bovine, pork, horse). On the same LOC used for DNA purification, we set up the on-chip PCR with real-time detection. Over 1,000 beef genomes, up to 0.01 horse or pork genomes were successfully detected in binary mixtures of pre-purified DNA and similarly, up to 0.01 % parts of exogenous meat were detected in binary mixtures of meat homogenates. The successful on-chip detection of exogenous DNA is a promising step toward the production of an effective microdevice for rapid, sensitive, and reliable identification of meat adulteration.

MicroRNAs (miRNAs) are endogenous, small (18–24 nt), non-coding RNAs that regulate gene expression. Among miRNAs, those bound to the AGO2 protein are the functionally active fraction which mediates the cell regulatory processes and regulate messages exchanged by cells. Several methods have been developed to purify this fraction of microRNAs, such as immunoprecipitation and immunoprecipitation-derived techniques. However, all these techniques are generally recognized as technically complicated and time consuming. Here, a new bio-functional surface for the specific capture of AGO2-bound microRNAs is proposed. Starting from a silicon oxide surface, a protein A layer was covalently bound via epoxy chemistry to orient specific anti-AGO2 antibodies on the surface. The anti-AGO2 antibodies captured the AGO2 protein present in cell lysate and in human plasma. The AGO2-bound microRNAs were then released by enzymatic digestion and detected via RT-qPCR. Control surfaces were also prepared and tested. Every step in the preparation of the bio-functional surfaces was fully characterized from the chemical, morphological and functional point of view. The resulting bio-functional surface is able to specifically capture the AGO2-bound miRNAs from biologically-relevant samples, such as cell lysate and human plasma. These samples contain different proportions of AGO2-bound microRNAs, as reliably detected with the immunocapture method here proposed. This work opens new perspectives for a simple and faster method to isolate not only AGO2-bound microRNAs, but also the multiprotein complex containing AGO2 and miRNAs.

The translational machinery, i.e., the polysome or polyribosome, is one of the biggest and most complex cytoplasmic machineries in cells. Polysomes, formed by ribosomes, mRNAs, several proteins and non-coding RNAs, represent integrated platforms where translational controls take place. However, while the ribosome has been widely studied, the organization of polysomes is still lacking comprehensive understanding. Thus much effort is required in order to elucidate polysome organization and any novel mechanism of translational control that may be embedded. Atomic force microscopy (AFM) is a type of scanning probe microscopy that allows the acquisition of 3D images at nanoscale resolution. Compared to electron microscopy (EM) techniques, one of the main advantages of AFM is that it can acquire thousands of images both in air and in solution, enabling the sample to be maintained under near physiological conditions without any need for staining and fixing procedures. Here, a detailed protocol for the accurate purification of polysomes from mouse brain and their deposition on mica substrates is described. This protocol enables polysome imaging in air and liquid with AFM and their reconstruction as three-dimensional objects. Complementary to cryo-electron microscopy (cryo-EM), the proposed method can be conveniently used for systematically analyzing polysomes and studying their organization.

The Sol-gel route is a versatile method to fabricate multi-layer, dense and homogeneous ZnO thin films with a controlled thickness and defects for a memristive application. In this work, sol-gel derived multi-layer undoped and Al-doped ZnO thin films were prepared by a spin-coating technique on SiO2/Ti/Pt and sil ica glass substrates. The effect of both Al doping and curing conditions on the structural and morphological features of ZnO films was investigated by complementary techniques, including electron microscopy, atomic force microscopy, X-ray photoelectron spectroscopy, micro-Raman spectroscopy, and X-ray diffraction anal- ysis. Electrical measurements were performed on SiO2/Ti/Pt/ZnO/Pt(dishes) and SiO2/Ti/Pt/ZnO(Al)/Pt(dishes) fabricated memristive cells and preliminary current-voltage curves were acquired.

Herein, we report a memristive response from Pt/TiO2/Ta2O5/Pt stack thin films with low SET and RESET voltages, and resistance ratio of 103. For the first time, Pt/TiO2/Ta2O5 stack thin films were produced by sol-gel procedure. The morphology and elemental composition of Pt/TiO2/Ta2O5 stacks were studied by a set of complementary techniques, including scanning electron microscopy (SEM), field-emission electron microscopy (FE-SEM), atomic force microscopy (AFM), and X-ray photoelectron spectroscopy (XPS). The thickness of the material was estimated from the transmittance spectrum by Pointwise Unconstrained Optimization Approach (PUMA). Keywords: Memristive response, Tantalum pentoxide, Titanium dioxide, Sol-gel, Thin films

Hepatitis C virus (HCV) is one of the main causes of chronic liver disease worldwide. The diagnosis and monitoring of HCV infection is a crucial need in the clinical management. The conventional diagnostic technologies are challenged when trying to address molecular diagnostics, especially because they require a complex and time-consuming sample preparation phase. Here, a new concept based on surface functionalization was applied to viral RNA purification: first of all polydimethylsiloxane (PDMS) flat surfaces were modified to hold RNA adsorption. After a careful chemical and morphological analysis of the modified surfaces, the functionalization protocols giving the best RNA adsorbing surfaces were applied to PDMS microdevices. The functionalized microdevices were then used for RNA purification from HCV infected human plasma samples. RNA purification and RT were successfully performed in the same microdevice chamber, saving time of analysis, reagents, and labor. The PCR protocol for HCV cDNA amplification was also implemented in the microdevice, demonstrating that the entire process of HCV analysis, from plasma to molecular readout, could be performed on-chip. Not only HCV but also other microdevice-based viral RNA detection could therefore result in a successful Point-of-Care (POC) diagnostics for resource-limited settings.

Modern Lab-on-a-chip (LOC) platforms for genomic applications integrate several biological tasks in a single device. Combination of these processes into a single device minimizes sample loss and contamination problems as well as reducing analysis time and costs. Here we present a study of a microchip platform aimed at analyzing issues arising from the combination of different functions, such as DNA purification from blood, target amplification by PCR and DNA detection in a single silicon-based device. DNA purification is realized through two different strategies: 1) amine groups coating microchannel surfaces and 2) magnetic nanoparticles coated by chitosan. In the first strategy silicon/Pyrex microdevices coated with 3-aminopropyltriethoxysilane (APTES) or 3-2-(2-aminoethylamino)-ethylamino]-propyltrimethoxysilane (AEEA) were examined and their efficiency in human genomic DNA adsorption/desorption was evaluated. APTES treatment was the most suitable for the purification of a reasonable amount of DNA in a state suitable for the following PCR step. The second strategy has instead the main advantage of avoiding an elution step, since the DNA adsorbed on the magnetic nanoparticles can be used as PCR template. On-chip PCR was performed in a custom thermocycler, while the detection of PCR products was carried out by fluorescence reading. A complete genetic analysis was demonstrated on the monolithic silicon/Pyrex microchip, starting from less than 1 [Formula: see text]L of human whole blood and arriving at SNPs identification. The successful integration of DNA purification, amplification and detection on a single microdevice was proven without the need for biological passivation steps and possibly simplifying the realization of genomic detection devices.

Pore formation of cellular membranes is an ancient mechanism of bacterial pathogenesis that allows efficient damaging of target cells. Several mechanisms have been described, however, relatively little is known about the assembly and properties of pores. Listeriolysin O (LLO) is a pH-regulated cholesterol-dependent cytolysin from the intracellular pathogen Listeria monocytogenes, which forms transmembrane β-barrel pores. Here we report that the assembly of LLO pores is rapid and efficient irrespective of pH. While pore diameters at the membrane surface are comparable at either pH 5.5 or 7.4, the distribution of pore conductances is significantly pH-dependent. This is directed by the unique residue H311, which is also important for the conformational stability of the LLO monomer and the rate of pore formation. The functional pores exhibit variations in height profiles and can reconfigure significantly by merging to other full pores or arcs. Our results indicate significant plasticity of large β-barrel pores, controlled by environmental cues like pH.

The present generation of mechanical heart-valves prostheses offers significantly improved blood compatibility, combined with appropriate physical and mechanical properties and durability; however, thromboembolism persists as a crucial clinical complication because of the key role played by adsorbed plasma proteins and adherent platelets in determining hemocompatibility. In the present work human plasma protein adsorption and platelet adhesion and activation were evaluated on four materials, selected according to their different physico-chemical and morphological properties: pyrolytic carbon (PyC), titanium alloy Ti–6Al–4V (Ti), tissue culture polystyrene (TCPS) and polystyrene sterilized by γ-irradiation (StPS). Morphological and chemical properties of surfaces were assessed by Time of Flight Secondary Ion Mass Spectrometry (Tof-SIMS), X-ray Photoemission Spectroscopy (XPS), Atomic Force Microscopy (AFM) and Contact Angle (CA) analysis, while protein adsorption and platelet adhesion and activation were evaluated by colorimetric and immunofluorescence tests and Scanning Electron Microscopy (SEM), respectively. Among the analyzed materials, PyC induced the lowest level of protein adsorption and platelet adhesion and activation. In particular, immunofluorescence analysis of fibronectin (FN), fibrinogen (FG), von Willebrand factor (vWF), Hageman factor (FXII) and albumin (ALB) showed that PyC surface was characterized by high levels of FG adsorption, low levels of FN, ALB and vWF and the absence of FXII. Finally, when analyzing the biological response as a function of surface properties, it was found that protein adhesiveness increased with increasing contact angle values. To the contrary, platelet activation related to the total amount of adherent proteins was poor while cell activation was possibly dependent on the detailed composition of the underlying protein layer. Furthermore, differences in surface roughness of the examined materials did not seem to influence the biological response in terms of platelet activation and protein adsorption.