Functionalization of TiO2 sol-gel derived films for cell confinement.

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.

Prototyping a memristive-based device to analyze neuronal excitability.

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.

Cell transfer of information via miR-loaded exosomes: a biophysical approach.

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.

Bio-functional surfaces for the immunocapture of AGO2-bound microRNAs.

MicroRNAs (miRNAs) are endogenous, small (18-24nt), 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.

Smart detection of microRNAs through fluorescence enhancement on a photonic crystal.

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.

On-chip purification and detection of hepatitis C virus RNA from human plasma.

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.

Enhanced fluorescence detection of miRNA-16 on a photonic crystal.

We report a novel sensing method for fluorescence-labelled microRNAs (miRNAs) spotted on an all-dielectric photonic structure. Such a photonic structure provides an enhanced excitation and a directional beaming of the emitted fluorescence, resulting in a significant improvement of the overall signal collected. As a result, the Limit of Detection (LoD) is demonstrated to decrease by a factor of about 50. A compact read-out system allows a wide-field imaging-based detection, with little or no optical alignment issues, which makes this approach particularly interesting for further development for example in microarray-type bioassays.

Innovative microRNA purification based on surface properties modulation.

The increasing interest in circulating microRNAs (miRNAs) as potential non-invasive cancer biomarkers has prompted the rapid development of several extraction techniques. However, current methods lack standardization and are costly and labor intensive. In light of this, we developed a microRNA solid-phase extraction strategy based on charge and roughness modulation on substrate surfaces. PECVD treated silicon oxide (PECVD-SO) and thermally grown silicon oxide (TG-SO) surfaces were functionalized with positively charged 3-aminopropyltriethoxysilanes (APTES) and neutral poly(ethylene glycol) silanes (PEG-s) mixed in different proportions to modulate the density of net positive charges and the roughness of the substrate. Characterization of the surfaces was performed by atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and s-SDTB (sulfosuccinimidyl-4-o-(4,4-dimethoxytrityl) butyrate) assay in order to investigate the surface morphology and chemical composition, respectively. Adsorption and elution efficiency were assessed by fluorescence microscopy by means of synthetic fluorescently labeled microRNAs. We identified PECVD-SO functionalized with 0.1% APTES and 0.9% 21-24 units long PEG-s as a promising surface able to selectively bind microRNAs and release them in the presence of a basic buffer (pH=9) compatible with downstream analyses. MicroRNA integrity was assessed by reverse transcription and real-time PCR and confirmed by electrophoresis (Agilent 2100 Bioanalyzer), while binding competition from circulating DNA and proteins was excluded by fluorescence analyses and real-time PCR. On the contrary, total RNA slightly decreased miRNA adsorption. In conclusion, we showed an innovative and easy solid-state purification method for circulating miRNAs based on charge interaction, which could pave the path to future diagnostic and prognostic assays feasible as a routine test.

One-shot genetic analysis in monolithic Silicon/Pyrex microdevices.

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.

Vapor-phase self-assembled monolayers of aminosilane on plasma-activated silicon substrates.

Aminosilane self-assembled monolayers on silicon substrates have been prepared via a gas-phase procedure based on the consecutive reactions of the aminosilane precursor and water vapor. X-ray photoelectron spectroscopy, atomic force microscopy, and contact angle measurements have been used to characterize the aminosilane layers. For comparison, substrates modified with aminosilane through a liquid-phase procedure have been prepared and characterized by means of the same techniques. The vapor-based procedure was found to yield more uniform layers characterized by fewer and smaller aggregates as compared with liquid-treated substrates. Grazing angles reflection Fourier transform infrared measurements were carried out on the vapor-treated substrates before and after water exposure to investigate the hydrolysis of the alkoxy groups and further reaction to form siloxane bonds. The surface density of amino groups, as estimated through a colorimetric method, is very similar for vapor- and liquid-treated substrates, suggesting a similar reactivity and accessibility of the functional groups on the surface.

Immobilization of cationic rifampicin-loaded liposomes on polystyrene for drug-delivery applications.

Polymer-associated infections are a major problem in implanted or intravascular devices. Among others, microorganisms of the staphylococcal family have been identified as the most important culprit. Prevention of bacterial adhesion and colonization of polymeric surfaces by release of antimicrobial agents incorporated into the polymers itself are currently under study. We have developed a novel method for the functionalization of a polymeric surface which is based on the deposition of covalently coupled lipid structures from antibiotic loaded vesicles. We have found that such process significantly reduces the bacterial growth on polystyrene material. In this work, lipid coverage obtained from multilamellar (MLVs) and extruded unilamellar (LUVs) vesicles were analyzed with respect to their adhesion efficiency on three types of polystyrene (PS) well-plates. Two methods of lipid deposition were characterized and compared in terms of surface lipid density and time stability: deposition of cationic vesicles on negatively charged surfaces and formation of covalent linkages between functionalized lipids and amines enriched surfaces. In order to study the antibiotic encapsulation efficiency we measured how the rifampicin (RIF) loading was affected by changes of liposome charge upon introduction of various amounts of stearylamine (SA), distearoyl-trimethylammonium propane (DSTAP) or dioleoyloxypropyl-trimethylammonium chloride (DOTAP) into the liposomal formulation. RIF-coated polymeric surfaces were also tested against a Staphylococcus epidermidis strain to evaluate their efficacy in vitro, showing that only approximately 2% of such bacteria inoculated on MLV-treated PS substrate were able to proliferate. Covalently immobilized lipid films showed about a tenfold increase in time stability compared to electrostatically bonded lipid films. Furthermore, substrates covalently modified with RIF-loaded MLVs retained an antibacterial activity for up to 12 days when aged in buffer at 37 degrees C. Such antimicrobial polymer coatings show promise for their use as antibacterial barrier for the prevention of catheter-related infections.

Role of chemical interactions in bacterial adhesion to polymer surfaces.

Development of biomaterial-related infections is attracting an increasing interest due to the significant percentage of implant failure in the hospital care. Recent literature puts in evidence the dependence of the infection risk on the different biomaterials used, because of the different interactions between material surface and micro-organisms. Despite this, the mechanisms underlying the adhesion of bacteria to the biomaterial surface are still unclear. Aim of this work is to study the initial events of the processes responsible for the bacterial adhesion on polymers in order to prevent the development of bacterial infections and the consequent failure and replacement of biomedical devices. Electrostatic and Lifshitz-van der Waals forces are usually considered responsible for the interactions at the biomaterial interface. A new term that involves Lewis acid-base interactions is here introduced to better describe the bacterial adhesion to the polymer surface. Two requirements are needed to test this hypothesis: the development of an ideal polymeric surface in terms of chemical and morphological properties and the choice of a specific bacterial strain to be utilized as "probe". Experiments were worked out using an Escherichia coli (Gram-) strain that represent one of the principal isolates from infected biomaterial implants and its adhesion was investigated on polymers having different acid/basic character. The findings indicate that the bacterial adhesion is influenced by the chemical properties of the polymeric surface. These results may be interpreted taking into account a mechanism in which the acid/base (Lewis) interaction plays an important role.