Acidification of eukaryotic cell compartments is accomplished by vacuolar H+-ATPases (V-ATPases), large multisubunit complexes able to pump protons into the lumen of organelles or in the extracellular medium. V-ATPases are involved in a number of physiological cellular processes, and thus regulation of V-ATPase activity is of crucial importance for the cell. Indeed, dysfunction of V-ATPase or alterations of acidification have been recently recognized as key factors in a variety of human diseases. In this study, we applied capsule-based pH sensors and a real-time tracking method for investigating the role of the V1G1 subunit of V-ATPases in regulating the activity of the proton pump. We first constructed stable cell lines overexpressing or silencing the subunit V1G1. Second, we used fluorescent capsule-based pH sensors to monitor acidification before and during internalization by modified and control living cells. By using a simple real-time method for tracking capsule internalization, we were able to identify different capsule acidification levels with respect to each analyzed cell and to establish the kinetics for each. The intracellular pH measurements indicate a delay in acidification in either V1G1-overexpressing or V1G1-silenced cells compared to controls. Finally, in an independent set of experiments, we applied transmission electron microscopy and confocal fluorescence microscopy to further investigate the internalization of the capsules. Both analyses confirm that capsules are engulfed in acidic vesicular structures in modified and control cell lines. The use of capsule-based pH sensors allowed demonstration of the importance of the V1G1 subunit in V-ATPase activity concerning intravesicular acidification. We believe that the combined use of these pH-sensor system and such a real-time method for tracking their internalization path would contribute to systematically measure the proton concentration changes inside the endocytic compartments in various cell systems. This approach would provide fundamental information regarding molecular mechanisms and factors that regulate intracellular acidification, vesicular trafficking, and cytoskeletal reorganizations.

Carnosine is an endogenous dipeptide abundant in the central nervous system, where by acting as intracellular pH buffering molecule, Zn/Cu ion chelator, antioxidant and anti-crosslinking agent, it exerts a well-recognized multi-protective homeostatic function for neuronal and non-neuronal cells. Carnosine seems to counteract proteotoxicity and protein accumulation in neurodegenerative conditions, such as Alzheimer’s Disease (AD). However, its direct impact on the dynamics of AD-related fibril formation remains uninvestigated. We considered the effects of carnosine on the formation of fibrils/aggregates of the amyloidogenic peptide fragment Aβ1-42, a major hallmark of AD injury. Atomic force microscopy and thioflavin T assays showed inhibition of Aβ1-42 fibrillogenesis in vitro and differences in the aggregation state of Aβ1-42 small pre-fibrillar structures (monomers and small oligomers) in the presence of carnosine. in silico molecular docking supported the experimental data, calculating possible conformational carnosine/Aβ1-42 interactions. Overall, our results suggest an effective role of carnosine against Aβ1-42 aggregation.

In this work we report on the self-assembly of monodisperse iron oxide nanocrystals on silica-coated Au surfaces achieved by magnetic field-assisted solution deposition techniques and discuss the effects of the interactions that contribute to promote their ordered arrangement into small clusters, chain-like structures or high-density particle multilayer superlattices. The results highlight the roles of inter-particle and nanocrystal-substrate interactions in controlling the nucleation and growth of self-assembled clusters and superstructures made of spherical magnetic nanocrystals.

Here an EIS (electrochemical impedance spectroscopy) biochip to detect cell migration is demonstrated. This biochip has been inspired by a traditional transwell assay/modified Boyden chamber and consists of two compartments separated by a porous membrane. This structure (PDMS-based) is aligned to EIS sensors. Cells are seeded in the upper chamber through microfluidic channels. During migration cells go through the pores of the membrane and get in touch with the electrodes that detect migrated cells. The performance of our cell-chip was tested by investigating the migratory ability of hepatocellular carcinoma (HCC) cells as a function of microenvironment. For this purpose we challenged HCC cells to migrate on different extra-cellular matrix (ECM) components including laminin 1, collagen IV and laminin 5. The results reveal that our cell chip provides reliable results that consistently overlap with those obtained with traditional standardized Boyden chambers. Thus, we demonstrate a new, easy tool to study cell migration and to perform automatic assays. This approach is easier and faster than traditional transwell assays and can be suitable for high-throughput studies in drug discovery applications.

Graphene-based capsules have strong potential for a number of applications, including drug/gene delivery, tissue engineering, sensors, catalysis and reactors. The ability to integrate graphene into carrier systems with three-dimensional (3D) geometry may open new perspectives both for fundamental tests of graphene mechanics and for novel (bio)technological applications. However, the assembly of 3D complexes from graphene or its derivatives is challenging because of its poor stability under biological conditions. In this work, we attempted to integrate a layer of graphene oxide derivative into the shell of biodegradable capsules by exploiting a facile layer-by-layer (LbL) protocol. As a first step we optimized the LbL protocol to obtain colloidal suspensions of isolated capsules embedding the graphene oxide derivative. As a following step, we investigated in detail the morphological properties of the hybrid capsules, and how the graphene oxide derivative layer influences the porosity and the robustness of the multilayer composite shells. Finally, we verified the uptake of the capsules modified with the GO derivative by two cell lines and studied their intracellular localization and biocompatibility. As compared to pristine capsules, the graphene-modified capsules possess reduced porosity, reduced shell thickness and a higher stability against osmotic pressure. They show remarkable biocompatibility towards the tested cells and long-term colloidal stability and dispersion. By combining the excellent mechanical properties of a graphene oxide derivative with the high versatility of the LbL method, robust and flexible biocompatible polymeric capsules with novel characteristics have been fabricated.

Polyelectrolyte multilayer (PEM) capsules engineered with active elements for targeting, labeling, sensing and delivery hold great promise for the controlled delivery of drugs and the development of new sensing platforms. PEMcapsules composed of biodegradable polyelectrolytes are fabricated for intracellular delivery of encapsulated cargo (for example peptides, enzymes, DNA, and drugs) through gradual biodegradation of the shell components. PEM capsules with shells responsive to environmental or physical stimuli are exploited to control drug release. In the presence of appropriate triggers (e.g., pH variation or light irradiation) the pores of the multilayer shell are unlocked, leading to the controlled release of encapsulated cargos. By loading sensing elements in the capsules interior, PEM capsules sensitive to biological analytes, such as ions and metabolites, are assembled and used to detect analyte concentration changes in the surrounding environment. This Review aims to evaluate the current state of PEM capsules for drug delivery and sensing applications.

Liquid flow in microchannels is completely laminar and uniaxial, with a very low Reynolds number regime and long mixing lengths. To increase fluid mixing and solubility of reactants, as well as to reduce reaction time, complex three-dimensional networks inducing chaotic advection have to be designed. Alternatively, turbulence in the liquid can be generated by active mixing methods (magnetic, acoustic waves, etc.) or adding small quantities of elastic materials to the working liquid. Here, polyelectrolyte multilayer capsules embodying a catalytic polyoxometalate complex have been suspended in an aqueous solution and used to create elastic turbulence and to propel fluids inside microchannels as an alternative to viscoelastic polymers. The overall effect is enhanced and controlled by feeding the polyoxometalate-modified capsules with hydrogen peroxide, H2O2, thus triggering an on-demand propulsion due to oxygen evolution resulting from H2O2 decomposition. The quantification of the process is done by analysing some structural parameters of motion such as speed, pressure, viscosity, and Reynolds and Weissenberg numbers, directly obtained from the capillary dynamics of the aqueous mixtures with different concentrations of H2O2. The increases in fluid speed as well as the capsule-induced turbulence effects are proportional to the H2O2 added and therefore dependent on the kinetics of H2O2 dismutation.

Multicompartment, spherical microcontainers were engineered through a layer-by-layer polyelectrolyte deposition around a fluorescent core while integrating a ruthenium polyoxometalate (Ru4POM), as molecular motor, vis-a-vis its oxygenic, propeller effect, fuelled upon H2O2 decomposition. The resulting chemomechanical system, with average speeds of up to 25 mu ms(-1), is amenable for integration into a microfluidic set-up for mixing and displacement of liquids, whereby the propulsion force and the resulting velocity regime can be modulated upon H2O2-controlled addition.

Cell culture technologies were initially developed as research tools for studying cell functions, but nowadays they are essential for the biotechnology industry, with rapidly expanding applications requiring more and more advancements with respect to traditional tools. Miniaturization and integration of sensors and microfluidic components with cell culture techniques open the way to the development of cellomics as a new field of research targeting innovative analytic platforms for high-throughput studies. This approach enables advanced cell studies under controllable conditions by providing inexpensive, easy-tooperate devices. Thanks to their numerous advantages cell-chips have become a hotspot in biosensors and bioelectronics fields and have been applied to very different fields. In this review exemplary applications will be discussed, for cell counting and detection, cytotoxicity assays, migration assays and stem cell studies.

Parkinson’s disease (PD) is one of the most common neurodegenerative diseases. Genes which have been implicated in autosomal-recessive PD include PARK2 which codes for parkin, an E3 ubiquitin ligase that participates in a variety of cellular activities. In this study, we compared parkin-mutant primary fibroblasts, from a patient with parkin compound heterozygous mutations, to healthy control cells. Western blot analysis of proteins obtained from patient’s fibroblasts showed quantitative differences of many proteins involved in the cytoskeleton organization with respect to control cells. These molecular alterations are accompanied by changes in the organization of actin stress fibers and biomechanical properties, as revealed by confocal laser scanning microscopy and atomic force microscopy. In particular, parkin deficiency is associated with a significant increase of Young’s modulus of null-cells in comparison to normal fibroblasts. The current study proposes that parkin influences the spatial organization of actin filaments, the shape of human fibroblasts, and their elastic response to an external applied force.

Three-dimensional (3D) porous scaffolds based on collagen are promising candidates for soft tissue engineering applications. The addition of stimuli-responsive carriers (nano- and microparticles) in the current approaches to tissue reconstruction and repair brings about novel challenges in the design and conception of carrier-integrated polymer scaffolds. In this study, a facile method was developed to functionalize 3D collagen porous scaffolds with biodegradable multilayer microcapsules. The effects of the capsule charge as well as the influence of the functionalization methods on the binding efficiency to the scaffolds were studied. It was found that the binding of cationic microcapsules was higher than that of anionic ones, and application of vacuum during scaffolds functionalization significantly hindered the attachment of the microcapsules to the collagen matrix. The physical properties of microcapsules-integrated scaffolds were compared to pristine scaffolds. The modified scaffolds showed swelling ratios, weight losses and mechanical properties similar to those of unmodified scaffolds. Finally, in vitro diffusional tests proved that the collagen scaffolds could stably retain the microcapsules over long incubation time in Tris-HCl buffer at 37°C without undergoing morphological changes, thus confirming their suitability for tissue engineering applications. The obtained results indicate that by tuning the charge of the microcapsules and by varying the fabrication conditions, collagen scaffolds patterned with high or low number of microcapsules can be obtained, and that the microcapsules-integrated scaffolds fully retain their original physical properties.

We report on the first dielectric investigation of high-k yttrium copper titanate thin films, which were demonstrated to be very promising for nanoelectronics applications. The dielectric constant of these films is found to vary from 100 down to 24 (at 100 kHz) as a function of deposition conditions, namely oxygen pressure and film thickness. The physical origin of such variation was investigated in the framework of universal dielectric response and Cole–Cole relations and by means of voltage dependence studies of the dielectric constant. Surface-related effects and charge hopping polarization processes, strictly dependent on the film microstructure, are suggested to be mainly responsible for the observed dielectric response. In particular, the bulky behaviour of thick films deposited at lower oxygen pressure evolves towards a more complex and electrically heterogeneous structure when either the thickness decreases down to 50 nmor the films are grown under high oxygen pressure.

In this review we will overview novel nanotechnological nanocarrier systems for cancer therapy focusing on recent development in polyelectrolyte capsules for targeted delivery of antineoplastic drugs against cancer cells. Biodegradable polyelectrolyte microcapsules (PMCs) are supramolecular assemblies of particular interest for therapeutic purposes, as they can be enzymatically degraded into viable cells, under physiological conditions. Incorporation of small bioactive molecules into nano-to-microscale delivery systems may increase drug's bioavailability and therapeutic efficacy at single cell level giving desirable targeted therapy. Layer-by- layer (LbL) self-assembled PMCs are efficient microcarriers that maximize drug's exposure enhancing antitumor activity of neoplastic drug in cancer cells. They can be envisaged as novel multifunctional carriers for resistant or relapsed patients or for reducing dose escalation in clinical settings

A flow-injection impedimetric immunosensor for the sensitive, direct and label-free detection of cholera toxin is reported. A limit of detection smaller than 10 pM was achieved, a value thousands of times lower than the lethal dose. The developed chips fulfil the requirement of low cost and quick reply of the assay and are expected to enable field screening, prompt diagnosis and medical intervention without the need of specialized personnel and expensive equipment, a perspective of special relevance for use in developing countries. Since the chip layout includes two sensing areas each one with a 2 × 2 sensor array, our biochips can allow statistical or (alternatively) multiplex analysis of biorecognition events between antibodies immobilized on each working electrode and different antigens flowing into the chamber.

Glass micromachining is a basic technology to achieve microfluidic networks for lab-on-a-chip applications. Among several methods to microstructure glass, the simplest and most widely applied is wet chemical etching (WE). However, accurate control of the reaction conditions to perform reproducible, fast and safe glass etching is not straightforward. Herein, microwave-assisted WE is demonstrated to intensify the glass etching action under safe working and finely monitored operative conditions and to produce smooth deep channels in short processing times with reduced underetching effects.

AIM: The lack of sensitivity of chronic myeloid leukemia (CML) stem cells to imatinib mesylate (IM) commonly leads to drug dose escalation or early disease relapses when therapy is stopped. Here, we report that packaging of IM into a biodegradable carrier based on polyelectrolyte microcapsules increases drug retention and antitumor activity in CML stem cells, also improving the ex vivo purging of malignant progenitors from patient autografts. MATERIALS & METHODS: Microparticles/capsules were obtained by layer-by-layer (LbL) self-assembly of oppositely charged polyelectrolyte multilayers on removable calcium carbonate (CaCO(3)) templates and loaded with or without IM. A leukemic cell line (KU812) and CD34(+) cells freshly isolated from healthy donors or CML patients were tested. RESULTS & DISCUSSION: Polyelectrolyte microcapsules (PMCs) with an average diameter of 3 microm, fluorescently labelled multilayers sensitive to the action of intracellular proteases and 95-99% encapsulation efficiency of IM, were prepared. Cell uptake efficiency of such biodegradable carriers was quantified in KU812, leukemic and normal CD34(+) stem cells (range: 70-85%), and empty PMCs did not impact cell viability. IM-loaded PMCs selectively targeted CML cells, by promoting apoptosis at doses that exert only cytostatic effects by IM alone. More importantly, residual CML cells from patient leukapheresis products were reduced or eliminated more efficiently by using IM-loaded PMCs compared with freely soluble IM, with a purging efficiency of several logs. No adverse effects on normal CD34(+) stem-cell survival and their clonogenic potential was noticed in long-term cultures of hematopoietic progenitors in vitro. CONCLUSION: This pilot study provides the proof-of-principle for the clinical application of biodegradable IM-loaded PMC as feasible, safe and effective ex vivo purging agents to target CML stem cells, in order to improve transplant outcome of resistant/relapsed patients or reduce IM dose escalation.

The sonication-assisted layer-by-layer (SLBL) technology was developed to combine necessary factors for an efficient drug-delivery system: (i) control of nanocolloid size within 100 - 300 nm, (ii) high drug content (70% wt), (iii) shell biocompatibility and biodegradability, (iv) sustained controlled release, and (v) multidrug-loaded system. Stable nanocolloids of Paclitaxel (PTX) and lapatinib were prepared by the SLBL method. In a multidrug-resistant (MDR) ovarian cancer cell line, OVCAR-3, lapatinib/PTX nanocolloids mediated an enhanced cell growth inhibition in comparison with the PTX-only treatment. A series of in vitro cell assays were used to test the efficacy of these formulations. The small size and functional versatility of these nanoparticles, combined with their ability to incorporate various drugs, indicates that lapatinib/PTX nanocolloids may have in vivo therapeutic applications.

Oxygen consumption rate (OCR) is a significant parameter helpful to determine in vitro respiratory efficiency of living cells. Oxygen is an excellent oxidant and its electrocatalytic reduction on a noble metal allows accurately detecting it. By means of microfabrication technologies, handy, low-cost, and disposable chip can be attained, minimizing working volumes and improving sensitivity and response time. In this respect, here is presented a microoxygraph device (MOD), based on Clark’s electrode principle, displaying many advantageous features in comparison to other systems. This lab-on-chip platform is composed of a three-microelectrode detector equipped with a microgrooved electrochemical cell, sealed with a polymeric reaction chamber. Au working/counter electrodes and Ag/AgCl reference electrode were fabricated on a glass slide. A microchannel was realized by photoresist lift-off technique and a polydimethylsiloxane (PDMS) nanoporous film was integrated as oxygen permeable membrane (OPM) between the probe and the microreaction chamber. Electrochemical measurements showed good reproducibility and average response time, assessed by periodic injection and suction of a reducing agent. OCR measurements on 3T3 cells, subjected, in real time, to chemical stress on the respiratory chain, were able to show that this chip allows performing consistent metabolic analysis.

We review the current research on nanodevices with nanoparticles which present unique challenges in both the realization of well-controlled interfaces at the nanoscale and the ability to adequately characterize their electrical properties. In particular, we discuss the fabrication and electrical characterization of such nanodevices with special attention to devices based on metal and magnetic nanoparticles.

The predicted 22-nm barrier which is seemingly going to put a final stop to Moore’s law is essentially related to the resolution limit of lithography. Consequently, finding suitable methods for fabricating and patterning nanodevices is the true challenge of tomorrow’s electronics. However, the pure matter of moulding devices and interconnections is interwoven with research on new materials, as well as architectural and computational paradigms. In fact, while the performance of any fabrication process is obviously related to the characteristic of the materials used, a particular fabrication technique can put constraints on the definable geometries and interconnection patterns, thus somehow biasing the upper levels of the computing machine. Further, novel technologies will have to account for heat dissipation, a particularly tricky problem at the nanoscale, which could in fact prevent the most performing nanodevice from being practically employed in complex networks. Finally, production costs – exponentially growing in the present Moore rush – will be a key factor in evaluating the feasibility of tomorrow technologies. The possible approaches to nanofabrication are commonly classified into top-down and bottom-up. The former involves carving small features into a suitable bulk material; in the latter, small objects assemble to form more complex and articulated structures. While the present technology of silicon has a chiefly top-down approach, bottom-up approaches are typical of the nanoscale world, being directly inspired by nature where molecules are assembled into supramolecular structures, up to tissues and organs. As top-down approaches are resolution-limited, boosting bottom-up approaches seems to be a good strategy to future nanoelectronics; however, it is highly unlikely that no patterning will be required at all, since even with molecular-scale technologies there is the need of electrically contacting the single elements and this most often happens through patterned metal contacts, although all-molecular devices were also proposed. Here, we will give some insight into both top-down and bottom-up without the intention to be exhaustive, because of space limitations.

Films of two fluorinated block copolymers (P(S81-Sz6) and P(S81-Sz11)) were investigated and compared with those of a commercial Teflon-like polymer (PTFE-AF) in the search for novel non-biofouling coatings able to minimize the amount of tear fluid (TF) proteins absorbed on contact lens (CL) surfaces. The adsorption of a solution containing lysozyme, albumin and immunoglobulin G (mimicking the TF composition) on the fluorinated block copolymers was evaluated using a quartz crystal microbalance. P(S81-Sz11) was found to resist protein adsorption more effectively than P(S81-Sz6) and PTFE-AF. The different interaction of P(S81-Sz6) and P(S81-Sz11) with the artificial TF was attributed to creation of a more heterogeneous and moderately hydrophobic surface of the latter polymer film by dynamic contact angle and atomic force microscopy studies. Moreover, deposition of P(S81-Sz11) on a CL-like silicone (PDMS) and a CL thin films demonstrated a protein adsorption reduction of up to 70% relative to pristine PDMS and commercial CL thin films.

Prostate cancer affects a large part of the western male population. The need for an early and accurate detection is thus a great challenge in common clinical practice, but the lack of specificity of the serum marker PSA (Prostate Specific Antigen) is a serious problem since its increased concentration can be related to several abnormalities. PSA, however, is found in serum in both a free and a complexed form with other proteins and the percentage amount of unbound PSA (the free-to-total PSA ratio) can be employed to distinguish prostate cancer from benign prostatic conditions, and also to predict the future risk of prostate cancer. To improve the operating characteristics of current PSA tests and to provide a clinical tool able to run label-free and sensitive analysis, we thus developed a biosensing platform based on Electrochemical Impedance Spectroscopy (EIS), which allows the contemporary detection of free and total PSA on a single biochip, enabling a quick screening for the risk of prostate cancer thanks to the presence of two different immobilized antibodies specific for the different antigens researched.

The aim of this work was the superficial activation, by means of plasma treatments, of crosslinked collagen-based scaffolds for nerve regeneration, in order to immobilize anionic and cationic microcapsules (MCPs) for drug delivery. Matrices with axially oriented pores have the potential to improve the regeneration of peripheral nerves and spinal cord by physically supporting and guiding the growth of neural structures across the site of injury. To improve mechanical resistance and stability in water solutions, it is necessary to crosslink collagenous fibres by formation of amide bonds with consequent reduction of free amino and carboxylic groups useful for immobilization approach of drug delivery systems like MCPs. Plasma chemical processes represent a successful approach because allow polar groups to be grafted on the surface, without modifying the massive properties of the bulk. Plasma surface modification was performed in a capacitively-coupled rf (13.56 MHz) glass reactor fed with different precursors like N2, H2O, C2H4 to study the effect of plasma parameters on the chemical properties of the resulting material and its ability to improve the immobilization of polyelectrolyte MCPs. Cylindrical scaffolds were synthesized by freeze-drying technique and dehydrothermally crosslinked. Polyelectrolyte capsules were obtained by LbL method. Scaffolds were characterized by means of WCA and XPS. Fluorescence microscopy was used to verify MCPs immobilization. After treatments, scaffolds became hydrophilic and able to absorb water. The success of grafting, on the external surface and within the scaffold core, was clearly revealed. The obtained results demonstrate that plasma processing of cross-linked collagen allows to enhance MCPs immobilization and that, by changing the typology of functional groups on the plasma treated surfaces, a different attitude to immobilize negatively or positively charged MCPs is observed.

The efficient internalization of TGF-beta inhibitor-loaded polyelectrolyte capsules and particles is studied in two HCC cell lines. Two polyelectrolyte pairs (biocompatible but not degradable and biodegradable crosslinked with gluteraldehyde) are employed for coating. The capsules are characterized by SEM. LY is successfully loaded inside the core and embedded between polymer layers. MS is used to quantify the loading efficiency by comparing post-loading and core-loading methods, since both coated templates and hollow shells are used as carriers. CLSM confirms dissolution of the pre-formed multilayer upon enzymatic degradation as the method of release, and migration assays demonstrate a higher inhibition efficiency of TGF-beta in tailored biodegradable capsules compared to free LY administration.

LY2157299 (LY), which is very small molecule bringing high cancer diffusion, is a pathway antagonist against TGFβ. LY dosage can be diluted by blood plasma, can be captured by immune system or it might be dissolved during digestion in gastrointestinal tract. The aim of our study is to optimize a "nano-elastic" carrier to avoid acidic pH of gastrointestinal tract, colon alkaline pH, and anti-immune recognition. Polygalacturonic acid (PgA) is not degradable in the gastrointestinal tract due to its insolubility at acidic pH. To avoid PgA solubility in the colon, we have designed its conjugation with Polyacrylic acid (PAA). PgA-PAA conjugation has enhanced their potential use for oral and injected dosage. Following these pre-requisites, novel polymeric nano-micelles derived from PgA-PAA conjugation and loading LY2157299 are developed and characterized. Efficacy, uptake and targeting against a hepatocellular carcinoma cell line (HLF) have also been demonstrated.

E-cadherin is the core protein of the epithelial adherens junction. Through its cytoplasmic domain, E-cadherin interacts with several signaling proteins; among them, - and -catenins mediate the linkof E-cadherin to the actin cytoskeleton. Loss of E-cadherin expression is a crucial step of epithelial-mesenchymal transition (EMT) and is involved in cancer invasion and metastatization. In human tumors,down-regulation of E-cadherin is frequently associated with poor prognosis. Despite the critical roleof E-cadherin in cancer progression, little is known about proteome alterations linked with its down-regulation. To address this point, we investigated proteomics, biophysical and functional changes ofepithelial breast cancer cell lines upon shRNA-mediated stable knockdown of E-cadherin expression(shEcad). shEcad cells showed a distinct proteomic signature including altered expression of enzymes andproteins involved in cytoskeletal dynamic and migration. Moreover, these results suggest that, besidestheir role in mechanical adhesion, loss of E-cadherin expression may contribute to cancer progressionby modifying a complex network of pathways that tightly regulate fundamental processes as oxidativestress, immune evasion and cell metabolism. Altogether, these results extend our knowledge on thecellular modifications associated with E-cadherin down-regulation in breast cancer cells.

Novel syntheticpeptidesrepresentsmartmoleculesforantigen–antibodyinteractionsinseveral bioanalyticsapplications,frompurificationtoserumscreening.Theirimmobilizationontoasolid phase isconsideredakeypointforsensitivityincreasing.Inthisview,weexploitedQuartzCrystal Microbalancewithsimultaneousfrequencyanddissipationmonitoring(QCM-D)withadoubleaim, specifically,asinvestigativetoolforspacersmonolayerassemblinganditsfunctionalevaluation,as well ashighsensitivemethodforspecificimmunosorbentassays.Themethodwasappliedto pancreaticductaladenocarcinoma(PDAC)detectionbystudyingtheinteractionsbetweensynthetic phosphorylatedandun-phosphorylated a-enolasepeptideswithseraofhealthyandPDACpatients.The syntheticpeptideswereimmobilizedonthegoldsurfaceoftheQCM-Dsensorviaaself-assembled alkanethiolmonolayer.Thepresentedexperimentalresultscanbeappliedtothedevelopmentof surfaceslesssensitivetonon-specificinteractionswiththefinaltargettosuggestspecificprotocolsfor detectingPDACmarkerswithun-labeledbiosensors.

In this study, the effects of Radio Electric Asymmetric Conveyer (REAC), a non-invasive physical treatment, on neuroinflammatory responses in a mouse model of parkinsonism induced by intoxication with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), were investigated in vivo. We found that the REAC tissue optimization treatment specific for neuro-regenerative purposes (REAC TO-RGN-N) attenuated the inflammatory picture evoked by MPTP-induced nigro-striatal damage in mice, decreasing the levels of pro-inflammatory molecules and increasing anti-inflammatory mediators. Besides, there was a significant reduction of both astrocyte and microglial activation in MPTP-treated mice exposed to REAC TO-RGN-N. These results indicated that REAC TO-RGN-N treatment modulates the pro-inflammatory responses and reduces neuronal damage in MPTP-induced parkinsonism.

Microfluidic technologies are gaining increasing importance due to their capability of manipulating fluids at the microscale that should allow to synthesize many products with surprisingly high yields and short reaction times. In the lab-on-chip field researchers have developed microfluidic apparatuses to provide special equipments for producing positron emission tomography (PET) radiopharmaceuticals in a quicker, safer, and more reliable way compared to traditional vessel-based approaches. In this paper, we have selected a number of polymeric materials, such as polydimethylsiloxane (PDMS), SU-8, and Teflon-like coatings deposited on PDMS or hard substrates, to be used for the fabrication of micro apparatuses for radiosynthesis. Their radioactivity resistance was investigated employing different setups and the results analyzed by atomic force microscopy (AFM), optical microscopy, and Fourier transform infrared spectroscopy (FT-IR). To evaluate undesired absorption effects in the investigated materials, the fluoride radioactive trapping inside microchannel was measured through autoradiography. We found out that polymeric materials such as SU-8 and Teflon coated on hard materials seem very appealing for fabricating microreactors for radiochemistry.

We have developed an integrated microfluidic platform for producing 2-[18F]-fluoro-2-deoxy-D-glucose (18F-FDG) in continuous flow from a single bolus of radioactive isotope solution, with constant product yields achieved throughout the operation that were comparable to those reported for commercially available vessel-based synthesisers (40–80%). The system would allow researchers to obtain radiopharmaceuticals in a dose-on-demand setting within a few minutes. The flexible architecture of the platform, based on a modular design, can potentially be applied to the synthesis of other radiotracers that require a two-step synthetic approach, and may be adaptable to more complex synthetic routes by implementing additional modules. It can therefore be employed for standard synthesis protocols as well as for research and development of new radiopharmaceuticals.

Increasing the ease and the rapidity of processing in micro and nanotechnology is an ongoing task, which is pursued in both the academic environment for investigation of novel systems and in industry for fabrication of complex circuits on a large scale. In the field of nanoelectronics, the major challenge is to demonstrate a feasible method for device implementation based on individual nanosize objects, such as nanowires, nanotubes and nanocrystals. However, integrating these small objects in a macroscopic circuit is a difficult task. So far, nanostructures have been wired by highly sophisticated techniques not suitable for large-scale integration in macroscopic circuits, such as electron-beam lithography or focused ion-beam deposition. Here we present a 'one-pot' and rapid approach to electrically interconnect individual nanowires from random spatial distribution, with high spatial and positioning resolution and a remarkable reduction in overall fabrication time with respect to the other expensive and laborious techniques. The reliability of such technique is demonstrated by implementing a single semiconductor nanowire device

A fundamental issue in biomedical and environmental sciences is the development of sensitive and robust sensors able to probe the analyte of interest, under physiological and pathological conditions or in environmental samples, and with very high spatial resolution. In this work, novel hybrid organic fibers that can effectively report the analyte concentration within the local microenvironment are reported. The nanostructured and flexible wires are prepared by embedding fluorescent pH sensors based on seminaphtho-rhodafluor-1-dextran conjugate. By adjusting capsule/polymer ratio and spinning conditions, the diameter of the fibers and the alignment of the reporting capsules are both tuned. The hybrid wires display excellent stability, high sensitivity, as well as reversible response, and their operation relies on effective diffusional kinetic coupling of the sensing regions and the embedding polymer matrix. These devices are believed to be a powerful new sensing platform for clinical diagnostics, bioassays and environmental monitoring.

An important goal of biomedical research is the development of tools for high throughput evaluation of drug effects and cytotoxicity tests. In this respect, electrochemical impedance spectroscopy (EIS) is an emerging technique for on-chip cell-based assays. For cell layers, RET (electron transfer resistence) and C (capacitance) are correlated to cell viability, adhesion and cytoskeleton organization and this approach has been successfully exploited to gain real-time information on cell behaviour. Here, for the first time, impedance based biochips in combination with complementary methodologies (including state-of-the-art AFM, viability test and western blot) are employed to perform a complete characterization of cell morphology and changes induced by copper ions on two cell lines (B104 and HeLa cells). Our results reveal a strong correlation between EIS data and both MTT test and AFM characterization. As a consequence, we expect that such on-chip assays can replace in vitro drug tests based on conventional biochemical methods, being very cheap and reusable and allowing to perform cytotoxicity tests without using any expensive reagent or equipment.

We report on the fabrication and single electron tunneling behaviour of large scale arrays of nanogap electrodes bridged by bisferrocene-gold nanoparticle hybrids (BFc-AuNP). Coulomb staircase was observed in the low temperature current-voltage curves measured on the junctions with asymmetric tunnel barriers. On the other hand, junctions with symmetric tunneling barrier exhibited mere nonlinear current voltage characteristics without discrete staircase. The experimental results agreed well with simulations based on the orthodox theory. The junction resistance showed thermally activated conduction behaviour at higher temperature. The overall voltage and temperature dependent results show that the transport behaviour of the large arrays of single particle devices obtained by a facile optical lithography and chemical etching process corresponds with the behaviour of single particle devices fabricated by other techniques like e-beam lithography and mechanical breaking methods.

To study the role of the solvent and of the laser fluence in the matrix-assisted pulsed laser evaporation (MAPLE) process, we used a soft polymer (polydimethylsiloxane— PDMS) as ‘‘sensing surface’’ and toluene as solvent. Thin films of the PDMS polymer were placed in the position of the growing film, while a frozen toluene target was irradiated with an ArF laser at the conventional fluences used in MAPLE depositions (60–250 mJ/cm2). Apart the absence of solute, the MAPLE typical experimental conditions for the deposition of thin organic layers were tested. The effects on the PDMS films of the toluene target ablation, at different fluences, were studied using atomic force microscopy and contact angles measurements. The results were compared with the effects produced on similar PDMS films by four different treatments (exposure to a drop of the solvent, to saturated toluene vapors and to plasma sources of two different powers). From this comparative study, it appears that depending on the MAPLE experimental conditions: (1) the MAPLE process may be ‘‘semidry’’ rather than purely dry (namely the solvent is likely to be present in the deposition environment near the growing film), (2) the solvent, if sufficiently volatile, is in form of vapor molecules (neutral, ionized and probably dissociated) rather than in liquid phase near the substrate and (3) at relatively high laser fluences ([150 mJ/cm2), the formation of an intense plasma plume results which can damage/affect a soft substrate as well as a growing polymer film.

Collagen, the major component of the extracellular matrix, key factor of tissue architecture, provides tensile strength, cell-matrix and matrix-matrix interactions. 2-D and 3-D collagen constructs are widely used as tissue scaffolds in a variety of biomedical applications. Here we synthetized scaffolds structured as thin films (30-50 μm in thickness) and we characterized them from a structural and functional point of view. Type I collagen isolated from bovine tendon (Sigma Aldrich) was suspended at 0.5% w/v in dilute hydrochloric acid (pH=3-3.2) by mixing at 15,000 rpm in an overhead blender, under proper refrigeration. After degassing via centrifugation, the slurry was cast in polystyrene molds and dried for at least 48 hours at room temperature, to obtain dry collagen films. In order to modulate their mechanical properties and degradation rate, the samples were subjected to two different crosslinking treatments, either dehydrothermal crosslinking (DHT) only, at 121°C for 24 hours, or DHT treatment combined with chemical crosslinking by means of a water soluble carbodiimide (DHT/EDC). First, atomic force microscopy measurements of the films showed differences in structure reticulation. As expected, the DHT/EDC scaffold surface presented a more intricate fibrillar assembly, and a lower swelling degree after the first 24 hours (about 30% less). Once integrated into appositely fabricated polymeric devices, DHT and DHT/EDC scaffolds were tested in cellular oxygen consumption and proliferation assays. Fibroblasts, seeded at the same densities on both DHT and DHT/EDC substrates, displayed different oxygen consumption rate (OCR) within 48 hours, reflecting dissimilarities in terms of structural organization and oxygen diffusion efficiency. The obtained results could represent a useful approach to indicate some culture parameters, such as cell-seeding optimal values for the feasibility of tissue models, depending on scaffold structure design.

Quantum-dot Cellular Automata (QCA) exploit quantum confinement, tunneling and electrostatic interaction for transistorless digital computing. Implementation at the molecular scale requires carefully tailored units which must obey several structural and functional constraints, ranging from the capability to confine charge efficiently on different 'quantum-dot centers' - in order to sharply encode the Boolean states - up to the possibility of having their state blanked out upon application of an external signal. In addition, the molecular units must preserve their geometry in the solid state, to interact electrostatically in a controlled way. Here, we present a novel class of organometallic molecules, 6-3,6-bis(1-ethylferrocen)-9H-carbazol-9-yl-6-hexan-1- thiols, which are engineered to satisfy all such crucial requirements at once, as confirmed by electrochemistry and scanning tunneling microscopy measurements, and first principles density functional calculations.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and lethal cancers in Europe and the United States. It has a very low 5 years-survival rate and its diagnosis is often late and imprecise due to the lack of specificity of currently used markers for PDAC. As previously demonstrated PDAC patients’ sera may contain autoantibodies towards phosphorylated a-enolase (ENOA), which in combination with other standard markers can increase specificity in diagnosis of PDAC. In this context we realized a microfluidic platform with integrated EIS biosensors. We achieved a specific antibodies detection by immobilizing onto electrodes peptides corresponding to a portion of ENOA. Phosphorylation of peptides was found to influence the recognition of antibodies in PDAC patients’ sera detected by the developed biochip thus validating the EIS technique as a strong tool for quick, cost-saving and label-free analysis of serum samples. Biochip results are in agreement with those from traditional techniques, such as ELISA and western blot, but measurements are much more sensitive and specific, increasing the possibility of PDAC diagnosis. In addition this approach is faster and more reproducible compared to traditional techniques making the developed biochips ideal for a quick, cost-saving and label-free analysis of serum samples.

Magnetic tunnel junctions sandwiching a superlattice thin film of iron oxide nanocrystals (NCs) have been investigated. The transport was found to be controlled by Coulomb blockade and single-electron tunneling, already at room temperature. A good correlation was identified to hold between the tunnel magnetoresistance (TMR), the expected magnetic properties of the NC arrays, the charging energies evaluated from current−voltage curves, and the temperature dependence of the junction resistance. Notably, for the first time, a switching from negative to positive TMR was observed across the Verwey transition, with a strong enhancement of TMR at low temperatures.

An organic field-effect transistor (T) is described, which comprises: a pair of source and drain electrode (S, D) which are formed on an electrically insulating layer (INS) and which are adapted to operate as the source and the collector of a flow of electric charge-carriers in which the majority carriers are free holes, and a layer of organic conductive material (F) which is disposed between the electrodes (S, D) for the formation of a conduction region through which the flow of charge carriers passes and the resistance of which is modulated by the application of a control voltage to a gate electrode (G) insulated from the conduction region, characterized in that the layer of organic conductive material (F) is a layer of biomolecular conductive material including a film of polypeptides or oligopeptides (P) .; The method for the manufacture of a transistor of this type comprises the formation of a film (F) of polypeptides or oligopeptides (P) on the electrically insulating layer (INS) in contact with the source and drain- electrodes (S, D) by immobilization of the polypeptides/oligopeptides (P) on the layer (INS) by deposition of a buffer solution of polypeptides/oligopeptides (P) , subsequent incubation for a predetermined period of time and at a predetermined temperature, removal of the buffer solution, and drying by exposure to a stream of gas.

A method and device, based on a film of a luminescent substance, such as colloidal semiconductor nanocrystals dispersed in a polymer matrix, for conducting quantitative and real-time analyses of PCR processes or of biomolecular interactions in genomics and/or proteomics. The optical detection system is based on FRET processes between the luminescent substance (which acts as the donor in the FRET process) and a suitable fluorophore (which acts as the acceptor species) with which the DNA or other biomolecule is marked. The device is essentially composed of a reaction microchamber with a wall formed by a thin film made of polymer material, in which the nanocrystals are uniformly dispersed, or made of a photoluminescent or electroluminescent polymer. Molecular probes are chemically immobilized on the surface of the polymer film for the specific recognition of the analyte which is to be determined in real time. The film of nanocrystals is excited by radiation at low wavelength (for example, UV/blue), and the radiation in the spectral emission window characteristic of the fluorescent marker of the biomolecule is detected. The specific photophysical characteristics of FRET processes make it possible to monitor in a selective way, in real time and in quantitative mode, the biomolecular interactions, which take place in the close proximity of the surface of the film (typically at distances of <10 nanometres), thus almost completely reducing possible interference caused by background signals and by the free biomolecules in solution (which have not interacted with the corresponding recognition sites). The characteristics of the device also enable simultaneous analyses to be conducted in parallel on different biomolecules (multiplexing).

A method of identifying target analytes in a sample, particularly a biological sample, comprising the steps of putting a plurality of target analytes, bound to a common luminescent marker, in contact with a plurality of molecular probes immobilized on a support, each of said molecular probes being capable of complementary binding to a respective target analyte, if said target analyte is present in the sample, providing to said support an excitation radiation and detecting an emission radiation coming from said support as a result of at least one complementary binding event, characterized by the fact that: said support comprises a plurality of photonic crystal resonators, at least two of said resonators being characterized by different resonance wavelengths; each of said molecular probes being fixed to a respective resonator;; and by the fact that the identification of at least one target analyte is carried out through the analysis of the total emission spectrum coming from said plurality of photonic crystals resonators, in order to detect possible wavelength resonance peaks generated as a result of the binding of said target analyte, bound to the common luminescent marker, to one or more of said molecular probes.

A chip for nucleic acid analysis includes a body (2, 9) , in which a detection chamber (7) is formed for accommodating nucleic acid probes (12, 12') . A waveguide (8) is integrated in the body (2, 9) is and is arranged at the bottom of the detection chamber (7) so that an evanescent wave (EW) , produced at an interface (8a) of the waveguide (8) when a light radiation is conveyed within the waveguide (8), is irradiated towards the inside of the detection chamber (7) . An apparatus for inspection of nucleic acid probes includes: a holder (22) , on which a chip (1) for nucleic acid analysis is loaded, the chip containing nucleic acid probes (12, 12'); a light source (24) for supplying an excitation radiation to the nucleic acid probes (12, 12'); and an optical sensor (25) arranged so as to receive radiation coming from the nucleic acid probes (12, 12') .

An optical apparatus for the inspection of nucleic acid probes includes: a holder (22) for housing a chip (1) for analysis of nucleic acids, containing nucleic acid probes (12, 12'); a light (24), for supplying an excitation radiation (WE) to the holder (22); and an optical sensor (25) for detecting images (IMG) of the nucleic acid probes (12, 12'), when a chip (1) is housed in the holder (22). The light source (24) is configured for polarizing the excitation radiation (WE) according to a excitation polarization direction (DE). Furthermore, the apparatus is provided with a sensing polarizing filter (27), which is arranged so as to intercept a reflected portion (WR) of the excitation radiation (WE), directed towards the optical sensor (25). The sensing polarizing filter (27) has a direction of the sensing polarization (DS) transverse to the excitation polarization direction (DE).

The present invention refers to a micro culture device (5) to be inserted in a chamber (4) of an oxygraph (7). The micro culture device is divided into an upper microchamber (2) and a lower microchamber (3) by a semi-permeable nanoporous membrane (1), suitable for acting as adhering support for cells. The upper microchamber (2) presents at its upper side either an inlet (2.1) coaxially aligned with an oxygraph plunger precision bore (6), or a semi-permeable membrane. The lower microchamber (3) presents at its bottom side either an inlet (3.1) coaxially aligned with an oxygraph piston bore (8) or a semi-permeable membrane (3.2).