Marianna Barbalinardo

Organic ultra-thin film transistors with a liquid gate for extracellular stimulation and recording of electric activity of stem cell-derived neuronal networks

Electronic transducers of neuronal cellular activity are important devices in neuroscience and neurology. Organic field-effect transistors (OFETs) offer tailored surface chemistry, mechanical flexibility, and high sensitivity to electrostatic potential changes at device interfaces. These properties make them attractive for interfacing electronics with neural cells and performing extracellular recordings and stimulation of neuronal network activity. In this work we operate pentacene ultra-thin film (9 nm thick) transistors with a liquid gate both as transducers and electrical stimulators of neuronal network activity. These devices are highly sensitive to small potential changes in cell medium and exhibit sufficient stability under standard cell culture conditions for nine days. We show that murine neural stem cells can be adhered on top of functional devices without the need for an additional layer of cell-adhesive molecules, and then differentiated into neuronal networks. OFET response is monitored during the different phases of the neuronal differentiation process up to nine days. Only when stem cells are differentiated into neurons, it is possible to measure electrical signals in the OFET current following the stimulation. Due to the large sensing area of our device, which accommodates from hundreds to thousands of interconnected neurons, the OFET electrical signals arise from the collective electrophysiological response of the neuronal population. The maximum extracellular potential change in the cleft region adjacent to the transistor surface amounts to 350 μV. This demonstrates that pentacene ultra-thin film OFETs enable good cellular adhesion and efficient coupling of the ionic currents at the biological-organic semiconductor interface with the OFET current.

Neural cell alignment by patterning gradients of the extracellular matrix protein laminin

Anisotropic orientation and accurate positioning of neural cells is achieved by patterning stripes of the extracellular matrix protein laminin on the surface of polystyrene tissue culture dishes by micromoulding in capillaries (MIMICs). Laminin concentration decreases from the entrance of the channels in contact with the reservoir towards the end. Immunofluorescence analysis of laminin shows a decreasing gradient of concentration along the longitudinal direction of the stripes. The explanation is the superposition of diffusion and convection of the solute, the former dominating at length scales near the entrance (characteristic length around 50 μm), the latter further away (length scale in excess of 900 μm). These length scales are independent of the channel width explored from about 15 to 45 μm. Neural cells are randomly seeded and selectively adhere to the pattern, leaving the unpatterned areas depleted even upon 6 days of incubation. Cell alignment was assessed by the orientation of the long axis of the 4′,6-diamidino-2-phenylindole-stained nuclei. Samples on patterned the laminin area exhibit a large orientational order parameter. As control, cells on the unpatterned laminin film exhibit no preferential orientation. This implies that the anisotropy of laminin stripes is an effective chemical stimulus for cell recruiting and alignment.

Tough and adhesive nanostructured calcium phosphate thin films deposited by the pulsed plasma deposition method

Calcium phosphate (CaP) coatings are commonly employed to improve the bioactivity of bone and dental metal implants, due to the chemical–physical similarity with the mineral phase of bone, envisaged to enhance the integration with the surrounding bone tissue. However, growing concerns about the use of commercial thick CaP coatings, mainly related to their weak mechanical properties, poor interface strength and micro-structural inhomogeneity, are leading the investigation of new and alternative CaP deposition techniques. In this study the feasibility to deposit tough and well adherent CaP thin films by a novel Pulsed Plasma Deposition (PPD) method on Ti alloy was investigated. Microstructural, chemical and morphological properties of the coatings as well as the nano-mechanical properties and their adhesion to the Ti-alloy substrate were extensively characterized. In vitro biocompatibility was also preliminary assessed evaluating the adhesion and proliferation of primary mouse osteoblasts. As-deposited CaP films were amorphous and exhibited dense and uniform surface composed of sub-micrometric aggregated globular particles. Noteworthy, mechanical properties of as-deposited films were comparable to the ones of commercial plasma-sprayed coatings despite the significant difference of thickness (a few hundred nanometers vs. tens of micrometers). After the thermal annealing of the as deposited films at 600 °C for 1 h in air, a transformation from amorphous calcium phosphate to crystalline hydroxyapatite (HA) phase occurred. The mechanical properties as well as the adhesion to substrate of the annealed films strongly improved respect to those of the as deposited films, displaying interesting high hardness, elastic strain to failure and plastic deformation resistance values. Finally, biological in vitro tests indicated good biocompatibility of both as-deposited and annealed films, with this latter showing better cells adhesion and proliferation compared to the former.

Customizing Properties of β-Chitin in Squid Pen (Gladius) by Chemical Treatments

The squid pen (gladius) from the Loligo vulgaris was used for preparation of β-chitin materials characterized by different chemical, micro- and nano-structural properties that preserved, almost completely the macrostructural and the mechanical ones. The β-chitin materials obtained by alkaline treatment showed porosity, wettability and swelling that are a function of the duration of the treatment. Microscopic, spectroscopic and synchrotron X-ray diffraction techniques showed that the chemical environment of the N-acetyl groups of the β-chitin chains changes after the thermal alkaline treatment. As a consequence, the crystalline packing of the β-chitin is modified, due to the intercalation of water molecules between β-chitin sheets. Potential applications of these β-chitin materials range from the nanotechnology to the regenerative medicine. The use of gladii, which are waste products of the fishing industry, has also important environmental implications.

A Nanoscale Interface Promoting Molecular and Functional Differentiation of Neural Cells.

Potassium channels and aquaporins expressed by astrocytes are key players in the maintenance of cerebral homeostasis and in brain pathophysiologies. One major challenge in the study of astrocyte membrane channels in vitro, is that their expression pattern does not resemble the one observed in vivo. Nanostructured interfaces represent a significant resource to control the cellular behaviour and functionalities at micro and nanoscale as well as to generate novel and more reliable models to study astrocytes in vitro. However, the potential of nanotechnologies in the manipulation of astrocytes ion channels and aquaporins has never been previously reported. Hydrotalcite-like compounds (HTlc) are layered materials with increasing potential as biocompatible nanoscale interface. Here, we evaluate the effect of the interaction of HTlc nanoparticles films with primary rat neocortical astrocytes. We show that HTlc films are biocompatible and do not promote gliotic reaction, while favouring astrocytes differentiation by induction of F-actin fibre alignment and vinculin polarization. Western Blot, Immunofluorescence and patch-clamp revealed that differentiation was accompanied by molecular and functional up-regulation of both inward rectifying potassium channel Kir 4.1 and aquaporin 4, AQP4. The reported results pave the way to engineering novel in vitro models to study astrocytes in a in vivo like condition.

Selective Growth of α-Sexithiophene by Using Silicon Oxides Patterns

A process for fabricating ordered organic films on large area is presented. The process allows growing sexithiophene ultra-thin films at precise locations on patterned Si/SiOx substrates by driving the orientation of growth. This process combines the parallel local anodic oxidation of Si/SiOx substrates with the selective arrangement of molecular ultra-thin film. The former is used to fabricate silicon oxide arrays of parallel lines of 400 nm in width over an area of 1 cm2. Selective growth arises from the interplay between kinetic growth parameters and preferential interactions with the patterned surface. The result is an ultra-thin film of organic molecules that is conformal to the features of the fabricated motives.

Self-protective action in multicomponent fluorescent self-assembled monolayers

We report on the fabrication of self-protective self-assembled monolayers constituted by a highly fluorescent component and a linear alkyl chain. We demonstrate the formation of an interpenetrated molecular network in which the alkyl component acts as an ultra-thin protective layer able to effectively shield the functional component from several types of aqueous solution.

Selective electrochemical decomposition of outgrowths and nanopatterning in La0.7Sr0.3MnO3 perovskite thin films

The outgrowth formation in inorganic thin films is a dramatic problem that has limited the technological impact of many techniques and materials. Outgrowths are often themselves part of the films, but are detrimental for vertical junctions since they cause short-circuits or work as defects, compromising the reproducibility and in some cases the operation of the corresponding devices. The problem of outgrowth is particularly relevant in ablation-based methods and in some complex oxides, but is present in a large variety of systems and techniques. Here we propose an efficient local electrochemical method to selectively decompose the outgrowths of conductive oxide thin films by electrochemical decomposition, without altering the properties of the background film. The process is carried out using the same set-up as for local oxidation nanolithography, except for the sign of the voltage bias and it works at the nanoscale both as serial method using a scanning probe and as parallel method using conductive stamps. We demonstrated our process using La0.7Sr0.3MnO3 perovskite as a representative material but in principle it can be extended to many other conductive systems.

Protein Corona Mediated Uptake and Cytotoxicity of Silver Nanoparticles in Mouse Embryonic Fibroblast

Medical applications of nanoparticles (NPs) require understanding of their interactions with living systems in order to control their physiological response, such as cellular uptake and cytotoxicity. When NPs are exposed to biological fluids, the adsorption of extracellular proteins on the surface of NPs, creating the so-called protein corona, can critically affect their interactions with cells. Here, the effect of surface coating of silver nanoparticles (AgNPs) on the adsorption of serum proteins (SPs) and its consequence on cellular uptake and cytotoxicity in mouse embryonic fibroblasts are shown. In particular, citrate-capped AgNPs are internalized by cells and show a time- and dose-dependent toxicity, while the passivation of the NP surface with an oligo(ethylene glycol) (OEG)-alkanethiol drastically reduces their uptake and cytotoxicity. The exposure to growth media containing SPs reveals that citrate-capped AgNPs are promptly coated and stabilized by proteins, while the AgNPs resulting from capping with the OEG-alkanethiol are more resistant to adsorption of proteins onto their surface. Using NIH-3T3 cultured in serum-free, the key role of the adsorption of SPs onto surface of NPs is shown as only AgNPs with a preformed protein corona can be internalized by the cells and, consequently, carry out their inherent cytotoxic activity.

Fluid Mixing for Low‐Power ‘Digital Microfluidics’ Using Electroactive Molecular Monolayers

A switchable electrode, which relies on an indium-tin oxide conductive substrate coated with a self-assembled monolayer terminated with an anthraquinone group (AQ), is reported as an electrowetting system. AQ electrochemical features confer the capability of yielding a significant modulation of surface wettability as high as 26° when its redox state is switched. Hence, an array of planar electrodes for droplets actuation is fabricated and integrated in a microfluidic device to perform mixing and dispensing on sub-nanoliter scale. Vehiculation of cells across microfluidic compartments is made possible by taking full advantage of surface electrowetting in culture medium.