NOVEL PLATFORM FOR BIOSENSING APPLICATION BASED ON CLUSTER-ASSEMBLED MATERIALS

Rapid methods to identify bacteria in biological samples are important for prompt antimicrobial therapy. The current detection methods are classical biological sample cultures and biochemical tests, which are however, time-consuming and not highly sensitive. A novel and highly performing approach is offered by aptamers acting as recognition elements able to detect epitopes on the surface of a bacterium. Aptamers interacting with specific bacteria are known and then could provide a solid base for developing promising solutions for this issue. With this PhD work I intended to tackle one drawback of aptamer-based biosensor: the lack of platforms for high density aptamers immobilization. Cluster-assembled thin films, have been optimized as supports to demonstrate that aptamers, targeting Staphylococcus aureus, well adhere on these substrates and keep their functionality. Moreover, the characteristics of the nanostructured zirconium oxide thin film: thermal stability, good reactivity towards -OH and -COOH groups and nano-morphology, make this material a suitable candidate for the realization of platforms for general screening and biosensing applications. This strategy will offer a promising way for the development of an user-friendly aptamer-based biosensors for screening biological samples. Furthermore, I focused on a technological problem, regarding the need of substrates to perform correlative light-electron microscopy(CLEM), designing, developing and testing a device which improve the feasibility of correlative fluorescence/confocal and scanning electron microscopy

Stiffening of nanoporous gold: experiment, simulation and theory

By combining electron microscopy measurements, atomistic simulations and elastic homogenization theory, we theoretically investigate the Young's modulus of nanoporous Au structures. Based on atomistic replicas generated starting from experimental tomographic evidence, atomistic simulations reveal that nanoporous Au stiffens as ligaments become finer, reproducing experimental findings obtained by nanoindentation of dealloyed samples. We argue that such a stiffening is neither due to surface stress nor to grain boundaries. Instead, we observe a direct quantitative correlation between the density of dislocations found in the material phase of the nanoporous structures and their Young's modulus and we propose a microscopic explanation of the observed stiffening. In particular, we show that local stress and strain fields in the neighborhood of dislocation cores allow dislocations to work as reinforcing solutes

Mesoporous Strontium-Doped Phosphate-Based Sol-Gel Glasses for Biomedical Applications

Mesoporous phosphate-based glasses have great potential as biomedical materials being able to simultaneously induce tissue regeneration and controlled release of therapeutic molecules. In the present study, a series of mesoporous phosphate-based glasses in the P2O5-CaO-Na2O system doped with 1, 3, and 5 mol % of Sr2+ were prepared using the sol-gel method combined with supramolecular templating. A sample without strontium addition was prepared for comparison. The non-ionic triblock copolymer EO20PO70EO20 (P123) was used as a templating agent. SEM images revealed that all synthesized glasses have an extended porous structure. This was confirmed by N2 adsorption-desorption analysis at 77 K that shows a porosity typical of mesoporous materials. 31P magic angle spinning nuclear magnetic resonance (31P MAS-NMR) and Fourier transform infrared (FTIR) spectroscopies have shown that the glasses are mainly formed by Q1 and Q2 phosphate groups. Degradation of the glasses in deionized water assessed over a 7-day period shows that phosphate, Ca2+, Na+ and Sr2+ ions can be released in a controlled matter over time. In particular, a direct correlation between strontium content and degradation rate was observed. This study shows that Sr-doped mesoporous phosphate-based glasses have great potential in bone tissue regeneration as materials for controlled delivery of therapeutic ions

Fabrication of nanoporous al by vapor-phase dealloying: Morphology features, mechanical properties and model predictions

The physical and chemical properties shown by nanoporous metals, related to their unique structure, make them very promising for application in several fields. Recently, vapor-phase dealloying has been reported as a method for the preparation of several non-noble nanoporous metals, alternatively to dealloying in aqueous solutions. Using this approach, we have successfully fabricated nanoporous Al starting from an Al20Zn80 nanocomposite obtained by ball milling. The nanocomposite was annealed at 550◦C under high-vacuum conditions, and the difference in the vapor pressures allowed the selective removal of Zn by vapor-phase dealloying. The morphology of the resulting nanoporous material was analyzed by Scanning Electron Microscopy showing pores from few to thousands of nm; moreover, the nanoporous 3D structure was observed through Serial Block Face-Scanning Electron Microscopy. A specific surface area as high as 73 m2 g−1 was estimated by N2 physisorption measurements. In addition, a fractal model able to well reproduce the morphology of nanoporous Al was built. This model has been used for predicting mechanical properties which are in good agreement with experimental data obtained by nanoindentation

Customized patterned substrates for highly versatile correlative light-scanning electron microscopy

Correlative light electron microscopy (CLEM) combines the advantages of light and electron microscopy, thus making it possible to follow dynamic events in living cells at nanometre resolution. Various CLEM approaches and devices have been developed, each of which has its own advantages and technical challenges. We here describe our customized patterned glass substrates, which improve the feasibility of correlative fluorescence/confocal and scanning electron microscopy

On-chip measurement of the Brownian relaxation frequency of magnetic beads using magnetic tunneling junctions

We demonstrate the detection of the Brownian relaxation frequency of 250 nm diameter magnetic beads using a lab-on-chip platform based on current lines for exciting the beads with alternating magnetic fields and highly sensitive magnetic tunnel junction MTJ sensors with a superparamagnetic free layer. The first harmonic out-of-phase component of the MTJ response gives the imaginary part of the magnetic bead susceptibility, which peaks at the Brownian relaxation frequency. This work paves the way to on-chip implementation of Brownian magnetorelaxometry in innovative “lab-on-a-bead” assays for biomolecular recognition

ROS and Lipid Droplet accumulation induced by high glucose exposure in healthy colon and Colorectal Cancer Stem Cells

Lipid Droplets (LDs) are emerging as crucial players in colon cancer development and maintenance. Their expression has been associated with high tumorigenicity in Cancer Stem Cells (CSCs), so that they have been proposed as a new functional marker in Colorectal Cancer Stem Cells (CR-CSCs). They are also indirectly involved in the modulation of the tumor microenvironment through the production of pro-inflammatory molecules. There is growing evidence that a possible connection between metabolic alterations and malignant transformation exists, although the effects of nutrients, primarily glucose, on the CSC behavior are still mostly unexplored. Glucose is an essential fuel for cancer cells, and the connections with LDs in the healthy and CSC populations merit to be more deeply investigated. Here, we showed that a high glucose concentration activated the PI3K/AKT pathway and increased the expression of CD133 and CD44v6 CSC markers. Additionally, glucose was responsible for the increased amount of Reactive Oxygen Species (ROS) and LDs in both healthy and CR-CSC samples. We also investigated the gene modulations following the HG treatment and found out that the healthy cell gene profile was the most affected. Lastly, Atorvastatin, a lipid-lowering drug, induced the highest mortality on CR-CSCs without affecting the healthy counterpart

Rational design of a user-friendly aptamer/peptide-based device for the detection of staphylococcus aureus

The urgent need to develop a detection system for Staphylococcus aureus, one of the most common causes of infection, is prompting research towards novel approaches and devices, with a particular focus on point-of-care analysis. Biosensors are promising systems to achieve this aim. We coupled the selectivity and affinity of aptamers, short nucleic acids sequences able to recognize specific epitopes on bacterial surface, immobilized at high density on a nanostructured zirconium dioxide surface, with the rational design of specifically interacting fluorescent peptides to assemble an easy-to-use detection device. We show that the displacement of fluorescent peptides upon the competitive binding of S. aureus to immobilized aptamers can be detected and quantified through fluorescence loss. This approach could be also applied to the detection of other bacterial species once aptamers interacting with specific antigens will be identified, allowing the development of a platform for easy detection of a pathogen without requiring access to a healthcare environment

Nitric oxide synthase mediates PC12 differentiation induced by the surface topography of nanostructured TiO2

Background: Substrate nanoscale topography influences cell proliferation and differentiation through mechanisms that are at present poorly understood. In particular the molecular mechanism through which cells 'sense' and adapt to the substrate and activate specific intracellular signals, influencing cells survival and behavior, remains to be clarified. Results: To characterize these processes at the molecular level we studied the differentiation of PC12 cells on nanostructured TiO2 films obtained by supersonic cluster beam deposition. Our findings indicate that, in PC12 cells grown without Nerve Growth Factor (NGF), the roughness of nanostructured TiO2 triggers neuritogenesis by activating the expression of nitric oxide synthase (NOS) and the phospho-extracellular signal-regulated kinase 1/2 (pERK1/2) signaling. Differentiation is associated with an increase in protein nitration as observed in PC12 cells grown on flat surfaces in the presence of NGF. We demonstrate that cell differentiation and protein nitration induced by topography are not specific for PC12 cells but can be regarded as generalized effects produced by the substrate on different neuronal-like cell types, as shown by growing the human neuroblastoma SH-SY5Y cell line on nanostructured TiO2. Conclusion: Our data provide the evidence that the nitric oxide (NO) signal cascade is involved in the differentiation process induced by nanotopography, adding new information on the mechanism and proteins involved in the neuritogenesis triggered by the surface properties