Simone Bonetti

A transparent organic transistor structure for bidirectional stimulation and recording of primary neurons

Real-time stimulation and recording of neural cell bioelectrical activity could provide an unprecedented insight in understanding the functions of the nervous system, and it is crucial for developing advanced in vitro drug screening approaches. Among organic materials, suitable candidates for cell interfacing can be found that combine long-term biocompatibility and mechanical flexibility. Here, we report on transparent organic cell stimulating and sensing transistors (O-CSTs), which provide bidirectional stimulation and recording of primary neurons. We demonstrate that the device enables depolarization and hyperpolarization of the primary neuron membrane potential. The transparency of the device also allows the optical imaging of the modulation of the neuron bioelectrical activity. The maximal amplitude-to-noise ratio of the extracellular recording achieved by the O-CST device exceeds that of a microelectrode array system on the same neuronal preparation by a factor of 16. Our organic cell stimulating and sensing device paves the way to a new generation of devices for stimulation, manipulation and recording of cell bioelectrical activity in vitro and in vivo.

A nanostructured conductive bio-composite of silk fibroin–single walled carbon nanotubes

Silk fibroin (SF), a protein core fibre from the silkworm Bombyx mori, has huge potential to become a sustainable, biocompatible, and biodegradable material platform that can pave the way towards the replacement of plastic in the fabrication of bio-derived materials for a variety of technological and biomedical applications. SF has remarkable mechanical flexibility, controllable biodegradability, biocompatibility and is capable of drug/doping inclusion, stabilization and release. However, the dielectric properties of SF limit its potential as a direct bioelectronic interface in biomedical devices intended to control the bioelectrical activity of the cell for regenerative purposes. In this work, a novel wet templating method is proposed to generate nanostructured, conductive Silk Fibroin (SF) composite films. We combine the unusual properties of SF, such as its mechanical properties, its convenience and biocompatibility with the electrical conductivity and stiffness of Single Walled Carbon Nanotubes (SWCNTs). The presented SF-SWCNT composite displays a periodic architecture where SWCNTs are regularly and homogeneously distributed in the SF protein matrix. The morphological and chemo-physical properties of the nanocomposite are analysed and defined by SEM, Raman Spectroscopy, ATR-IR, UFM and contact angle analyses. Notably, the SF-SWCNT composite film is conductive, showing additional functionality compared to the dielectric properties of the bare SF film. Finally, SF-SWCNT is biocompatible and enables the growth of primary rat Dorsal Root Ganglion (DRG) neurons. Collectively our results demonstrate that the nanostructured, conductive, robust and biocompatible SF-SWCNT composite can be fabricated using a wet templating method, paving the way towards the fabrication and development of silk-based electronic devices for use in bioelectronic and biomedical applications.

A lysinated thiophene-based semiconductor as a multifunctional neural bioorganic interface.

Lysinated molecular organic semiconductors are introduced as valuable multifunctional platforms for neural cells growth and interfacing. Cast films of quaterthiophene (T4) semiconductor covalently modified with lysine-end moieties (T4Lys) are fabricated and their stability, morphology, optical/electrical, and biocompatibility properties are characterized. T4Lys films exhibit fluorescence and electronic transport as generally observed for unsubstituted oligothiophenes combined to humidity-activated ionic conduction promoted by the charged lysine-end moieties. The Lys insertion in T4 enables adhesion of primary culture of rat dorsal root ganglion (DRG), which is not achievable by plating cells on T4. Notably, on T4Lys, the number on adhering neurons/area is higher and displays a twofold longer neurite length than neurons plated on glass coated with poly-l-lysine. Finally, by whole-cell patch-clamp, it is shown that the biofunctionality of neurons cultured on T4Lys is preserved. The present study introduces an innovative concept for organic material neural interface that combines optical and iono-electronic functionalities with improved biocompatibility and neuron affinity promoted by Lys linkage and the softness of organic semiconductors. Lysinated organic semiconductors could set the scene for the fabrication of simplified bioorganic devices geometry for cells bidirectional communication or optoelectronic control of neural cells biofunctionality.

Bio-doping of regenerated silk fibroin solution and films: a green route for biomanufacturing

Silk fibroin (SF) is a natural biocompatible material that can be integrated in a variety of photonic systems and optoelectronics: i.e. organic lasing from dye-doped nano-structured silk film. In this context, biological incorporation of doping molecules into SF by means of feeding silk worms with dyes to their diet could be an innovative and eco-sustainable approach to obtain doped SF substrates, thus avoiding additional chemical processes and post-treatments of the protein solution. In the present work, we demonstrated that SF regenerated solutions and films containing rhodamine B (RhB) could be successfully obtained from the cocoons of Bombyx mori fed with a RhB-added diet (RhB-md-SF). Comparative analyses of optical and vibration characteristics of the RhB-md-SF solution and films with those of white SF blended with RhB (RhB-d-SF) revealed significant differences, suggesting that the silkworms metabolism could be involved in the binding mechanism of SF with RhB. In conclusion, we observed that the doping diet is a promising method for the green fabrication of SF-based optically active materials, and it opens novel routes for silk-based biophotonics.

Effect of different fabrication methods on the chemo-physical properties of silk fibroin films and on their interaction with neural cells

In this study, we investigated the influence of processing methods on the chemo-physical properties of silk fibroin (SF) film and on their interaction with neural cells. Structural, thermal and morphological analysis revealed a strong correlation between the conformation, stability and texture of silk films and the fabrication conditions. An increase in temperature, methanol treatment and the use of a microfluidic approach led to an improvement in SF film stability in terms of β-sheet content, mechanical resistance, dissolution and enzymatic degradation. An effect on the interaction of SF films with neural cells, through a modulation of the surface properties, was also observed. In particular, hydrophobic surfaces induce proliferation of astrocytes and neuron adhesion whereas hydrophilic surfaces promote a remarkable neurite outgrowth. A detailed knowledge of the effect of manufacturing parameters on SF film properties can facilitate and extend the applications of silk-based biomaterials in tissue engineering and drug release systems.

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.

Silk fibroin film from Golden‐Yellow Bombyx mori is a biocomposite that contains lutein and promotes axonal growth of primary neurons

The use of doped silk fibroin (SF) films and substrates from Bombyx mori cocoons for green nanotechnology and biomedical applications has been recently highlighted. Cocoons from coloured strains of B. mori, such as Golden‐Yellow, contain high levels of pigments that could have a huge potential for the fabrication of SF based biomaterials targeted to photonics, optoelectronics and neuroregenerative medicine. However, the features of extracted and regenerated SF from cocoons of B. mori Golden‐Yellow strain have never been reported. Here we provide a chemophysical characterization of regenerated silk fibroin (RSF) fibers, solution, and films obtained from cocoons of a Golden‐Yellow strain of B. mori, by SEM, 1H‐NMR, HPLC, FT‐IR, Raman and UV‐Vis spectroscopy. We found that the extracted solution and films from B. mori Golden‐Yellow fibroin displayed typical Raman spectroscopic and optical features of carotenoids. HPLC‐analyses revealed that lutein was the carotenoid contained in the fiber and RSF biopolymer from yellow cocoons. Notably, primary neurons cultured on yellow SF displayed a threefold higher neurite length than those grown of white SF films. The results we report pave the way to expand the potential use of yellow SF in the field of neuroregenerative medicine and provide green chemistry approaches in biomedicine.

A self-assembled lysinated perylene diimide film as a multifunctional material for neural interfacing

We report the design, synthesis and structure-property investigation of a new perylene diimide material (PDI-Lys) bearing lysine end substituents. Water processed films of PDI-Lys were prepared and their self-assembly, morphology and electrical properties in both inert and air environments were theoretically and experimentally investigated. With the aim of evaluating the potential of PDI-Lys as a biocompatible and functional neural interface for organic bioelectronic applications, its electrochemical impedance as well as the adhesion and viability properties of primary neurons on the PDI-Lys films were studied. By combining theoretical calculations and electrical measurements we show that due to conversion between neutral and zwitterionic anions, the PDI-Lys film conductivity increased significantly upon passing from air to an inert atmosphere, reaching a maximum value of 6.3 S m-1. We also show that the PDI-Lys film allows neural cell adhesion and neuron differentiation and decreases up to 5 times the electrode/solution impedance in comparison to a naked gold electrode. The present study introduces an innovative, water processable conductive film usable in organic electronics and as a putative neural interface.

Integration of organic electrochemical transistors and immuno-affinity membranes for label-free detection of interleukin-6 in the physiological concentration range through antibody–antigen recognition

We demonstrate the label-free and selective detection of interleukin-6 (IL-6), a key cell-signaling molecule in biology and medicine, by integrating an OECT with an immuno-affinity regenerated cellulose membrane. The objective of the membrane is to increase the local concentration of IL-6 at the sensing electrode and, thereby, enhance the device response for concentrations falling within the physiological concentration range of cytokines. The OECT gate electrode is functionalized with an oligo(ethylene glycol)-terminated self-assembled alkanethiolate monolayer (SAM) for both the immobilization of anti IL-6 antibodies and the inhibition of non-specific biomolecule binding. The OECT gate/electrolyte interface is exploited for the selective detection of IL-6 through the monitoring of antigen-antibody binding events occurring at the gate electrode.

Selective Self-Assembled Monolayer to passivate organic cell stimulating and sensing transistor (OCSTs)

Self-Assembled Monolayers (SAMs) on gold were successfully implemented to passivate gold contact organic cell stimulating and sensing transistors. The results show the devices to be greatly insulated from water. This breakthrough is proposed as a technological improvement for bio-stimulation and bio-sensing organic field-effect devices.