Teses / dissertações sobre o tema "FISIC platform"

Étudier les collisions ion-atome et ion-ion permet d'appréhender les probabilités des processus électroniques tels que la capture, l'ionisation et/ou l'excitation en maîtrisant le nombre d'électrons initialement liés à chaque partenaire de la collision. En allant de l'étude d'un système à trois corps (les deux noyaux avec un seul électron) vers des systèmes plus complexes impliquant des électrons supplémentaires permet d'examiner des effets sur la dynamique globale des électrons et par conséquent sur les sections efficaces des processus élémentaires.Dans la section théorique de la thèse, des calculs de sections efficaces sont effectués pour des systèmes ion-atome et ion-ion contenant jusqu'à deux électrons. Le carbone, l'hydrogène, l'azote et l'hélium ont été choisis comme partenaires de collision. Cela est réalisé en utilisant une approximation semi-classique non perturbative, qui consiste à traiter le mouvement des ions de manière classique, tandis que la dynamique des électrons est décrite en utilisant la mécanique quantique. Les collisions sont simulées à l'aide d'un programme “collision solver”, à partir duquel des sections efficaces pour différents processus peuvent être extraites.La partie expérimentale de la thèse est dédiée au développement technique, aux tests et à la caractérisation de divers instruments essentiels pour la réalisation d'expériences précises de collision ion-atome et ion-ion. Deux sources d'ions et les lignes de faisceau correspondantes ont été installées pour réaliser des collisions ion-ion garantissant qu'une large gamme de conditions expérimentales et de types de collisions. Une chambre de collision, un jet gazeux, un spectromètre d'ions et son détecteur associé, un système de détection de rayons X ont été développés et caractérisées pour réaliser les premières études.Dans l'ensemble, cette thèse combine de nouveaux calculs théoriques pour les collisions ion-atome et ion-ion avec des avancées expérimentales vers une configuration capable d'explorer une large gamme de systèmes de collision. Cette double approche est très bénéfique pour améliorer la compréhension de la dynamique des électrons dans les collisions ion-matière. Ces connaissances sont essentielles tant pour la recherche fondamentale que pour les applications pratiques dans divers domaines scientifiques et technologiques, tels que les plasmas astrophysiques, la recherche sur la fusion par confinement inertiel ou encore l'hadronthérapie
Studying ion-atom and ion-ion collisions allows us to understand the probabilities of electronic processes such as capture, ionization, and/or excitation by controlling the number of electrons initially bound to each collision partner. By progressing from the study of a three-body system (the two nuclei with a single electron) to more complex systems involving additional electrons, we can examine the effects on the overall electron dynamics and consequently on the cross-sections of elementary processes.In the theoretical section of the thesis, cross-section calculations are performed for ion-atom and ion-ion systems containing up to two electrons. Carbon, hydrogen, nitrogen, helium and their respective ions have been chosen as collision partners. This is achieved using a semi-classical non-perturbative approach: the relative motion of the partners is treated classically, while the electron dynamics is described quantally. The collisions are simulated using a “collision solver” program, from which cross sections for different processes can be extracted.The experimental part of the thesis is dedicated to the technical development, rigorous testing, and thorough characterization of various instruments critical for conducting precise ion-atom and ion-ion collision experiments. Two ion sources and their respective beamlines were set up to perform ion ion collisions, ensuring a large range of possible experimental conditions and collision systems can be explored. A collision chamber, gaseous jet, an ion spectrometer and its associated detector, as well as an x-ray detection system were developed and characterized to perform the preliminary experiments.Overall, this thesis combines new theoretical calculations for ion-atom and ion-ion collisions with experimental advancements towards a set-up capable of exploring a wide range of collision systems. The dual approach is very beneficial for enhancing the understanding of electron dynamics in ion-matter collisions. This knowledge is essential for both fundamental research and practical applications in various scientific and technological fields, such as astrophysical plasma, inertial confinement fusion research or hadrontherapy

The use of nanocarriers for drug delivery and imaging purposes have highly increased in the last decades. Both hard and soft matter-based formulations can provide selective and efficient treatment in several administration routes. Indeed, the biocompatibility and the biodegradability of the formulations represent a key requirement in order to translate the in vitro studies into in vivo investigations. Therefore, lipids are a safe choice as building blocks to formulate a large variety of liquid crystalline architectures in water. Vesicles, hexosomes and cubosomes have been adopted as nanomedicine platforms providing excellent biological performances. However, several drawbacks may impact the application of these carriers: the poor stability in the physiological environment and the biodegradability of the stabilizing agent required to sterically stabilized the nanoparticles (NPs) are few examples. Given the importance these materials have acquired nowadays in the nanomedicine field, this thesis is devoted to investigating on the factors that can enhance the physico-chemical and biological performances of these nanoparticles for systemic and topical administration. Most of the formulations presented in this thesis were prepared using monoolein as building block, given its biocompatibility and lower cytotoxicity in comparison with other surfactants. However, the potential application of cell-derived nanoparticles known as nanoerythrosomes for medical imaging was also explored. Therefore, the thesis evaluated different approaches: (i) evaluation of the effect of various stabilizers (modified poloxamers, hemicellulose and polyphosphoesters) on monoolein-based cubosomes features, in order to formulate nanoparticles suitable for systemic administration. This investigation was focused on the physico-chemical (bulk and surface) characterization of the empty carriers and of those loaded with antioxidants or fluorophores suitable for in vitro imaging. Bioassays (viability and uptake experiments) were conducted in order to evaluate the biological performance of the differently stabilized cubosomes. (ii) the effect of permeation enhancers and edge activators on monoolein-based vesicles and hexosomes for topical administration. In vitro permeation tests were performed to show the efficacy of these carriers into overcoming the stratum corneum, the first layer of the skin, to deliver antioxidants. (iii) the potential role of nanoparticles derived from red blood cells, nanoerythrosomes, as personal medicine for application in optical imaging. Cross-linking and Click Chemistry were employed to decorate the surface of the nanoparticles and their emission properties in a physiological buffer were evaluate.

Il presente lavoro di tesi nasce come collaborazione tra il Laboratorio di Progettazione Elettronica e il Laboratorio di Microscopia a Fluorescenza del Dipartimento di Fisica e Astronomia dell' Università di Bologna. In particolare nasce dalla volontà di dotare il dipartimento di un apparato sperimentale in grado di svolgere studi sulla Galvanotassia, un fenomeno biologico consistente nella migrazione di cellule sottoposte a stimolazione elettrica. La Galvanotassia è nota da fine '800 ma non sono ancora chiari i meccanismi cellulari che la provocano. Una migliore comprensione di tale fenomeno potrebbe portare importanti sviluppi in ambito medico, sia diagnostici che terapeutici. Dalla letteratura a riguardo non è emersa l'esistenza di apparecchi elettronici di controllo che permettano lo studio della Galvanotassia e che possano essere duttili a seconda del tipo di esperimento che si voglia svolgere. Da qui l'idea di iniziare lo sviluppo di un dispositivo elettronico, che fosse riprogrammabile, a basso costo e facilmente trasportabile. La progettazione di questo dispositivo ha portato ad una prima fase di test e verifiche sperimentali che hanno permesso di migliorare e affinare la costruzione di uno strumento di misura e controllo dei parametri relativi alla Galvanotassia. Sono già stati programmati test futuri che porteranno ad una versione definitiva dell' apparecchiatura alla quale succederanno più approfondite ricerche sul fenomeno della Galvanotassia.

A multilayered dielectric structure, namely a one dimensional photonic crystal (1DPC), is proposed as a suitable platform for photon management, due to the low absorption of the dielectric materials. When properly designed, a 1DPC can sustain surface electromagnetic modes called Bloch Surface Waves (BSWs). In this PhD Thesis it is shown how light coupled to BSW can be focused or guided by means of ultrathin polymeric refractive structures directly patterned on the surface. Moreover, by patterning the surface with surface relief gratings, far-field radiation can be efficiently coupled to the surface modes, thus providing an enhanced electromagnetic field at the truncation interface of the 1DPC. By shaping the grating in a circular symmetry, light can be in-plane focused into a sub-wavelength spot. The same structure can be used to re-shape the radiation pattern of dipolar emitters. It is shown that an emitter lying on the surface of the 1DPC couples to the photonic structure and the fluorescence radiated couple with the surface modes. The so called BSW-coupled fluorescence propagates along the surface with low losses and a well-defined wavevector. By means of surface diffraction gratings properly designed, fluorescence can be extracted along any direction, thus improving the fluorescence collection with no need of high numerical aperture optics or critical alignements. A novel method for evaluating the enhancement gained with such photonic structures on the extraction efficiency is proposed. Such method is capable of providing at the same time spatial resolution, angular resolution and spectral resolution. A biosensing experiment to detect small amounts of labeled proteins is provided, in order to show the sensing capabilities of the photonic structure.

Early detection of cancer plays a crucial role in determining disease prognosis. The major challenge consists in the ability of identifying the disease through the quantification of a set of specific biomarkers in tissues and/or, more interestingly, released in the bloodstream. Non-invasiveness, sensitivity, parallelization, low cost, are some of the most relevant keywords in this field. Hence the development of miniaturized devices for the early detection of cancer is at the core of nanodiagnostics, requiring the recognition and quantification of low amounts of specific disease biomarkers, through the development of sensitive diagnostic tools. In this context, we have developed a nanodiagnostic platform for the non-invasive quantification of cancer biomarkers circulating in the bloodstream. The assay, that relies on Atomic Force Microscopy (AFM), is based on molecular manipulation to create density-optimized functional spots of surface-immobilized binders and differential AFM topography. It is label-free, allows the parallel detection of different cancer biomarkers, entails a single binder per antigen and when implemented with fluorescence labelling/readout can be used for epitope mapping. The possibility to exploit DNA nanografting and subsequent immobilization of binders through DNA-directed immobilization confers robustness to the assay. We explored the feasibility of novel binders as camelid nanobodies and aptamers, to improve the quality of the functionalization, and therefore device sensitivity, with the added advantage of binders easy engineering. In this study we focused on a prospective, clinically-relevant circulating cancer biomarker, the extra-cellular domain (ECD) of Human Epidermal Growth Factor Receptor (Her2), whose shedding and release in the blood is related to the progression of Her2-positive tumors and response to anticancer therapies. By employing robust, easily engineered camelid nanobodies as binders, we measured ECD-Her2 concentrations in the range of the actual clinical cutoff value for Her2 positive breast cancer. The specificity for Her2 detection was preserved when measured in complex matrices as standardized human serum, and in parallel with other potential biomarkers, demonstrating the intended implementation of multiplexing analysis, strongly required to define the biological tumor subtype and to univocally refer specific molecular levels to tumor status and progression. A better understanding of the Her2 receptor biology, overexpression in tumor cell membranes and release of the ECD to the bloodstream is however required to interpret the measured levels of ECD-Her2 at best. At present, there are controversial studies and conflicting results about the correlation between the protein levels in serum and the attested Her2 status in tumor tissue, which make the clinical significance of circulating ECD-Her2 still uncertain. Therefore we developed a multi-integrated approach in order to elucidate Her2 overexpression, dimerization and ECD shedding mechanism and to fully validate its prognostic value; moreover we preliminarily studied some fundamental aspects of the relationship between rafts-mediated exosomes formation and Her2 integration on them in order to clarify its possible role in metastasis occurrence. This approach relies on different multi-scale techniques and enables to correlate information coming from advanced optical microscopies (membrane proteins localization), nanotechnology-based diagnostic tools (detection of protein and vesicle biomarkers) and novel super resolution fluorescence microscopies (quantification and co-localization of different biomarkers). This innovative platform will be instrumental in identifying and quantifying clinically useful biomarkers and to translate basic science results into the clinics to impact cancer diagnosis, prognosis, and therapy.

In questa tesi magistrale ho studiato e analizzato gli strumenti e le risorse offerte dalla piattaforma di e-learning MOODLE e verificato quali opportunità offre per l'insegnamento e l'apprendimento. Ho effettuato, tenendo conto di queste risorse e di concetti di didattica della fisica, il trasferimento di uno dei corsi presenti sul sito ISHTAR dell'Università di Bologna, facendone la traduzione in inglese e integrandolo con elementi multimediali e forme di interfaccia, verifica e comunicazione proprie della piattaforma. Per un secondo corso, ho studiato e confrontato diverse metodologie di implementazione, basate su diversi obiettivi educativi.

2012/2013
Le proteine intrinsecamente disordinate (IDP), nello stato nativo non ripiegate, sono inclini all’aggregazione e direttamente correlate con lo sviluppo di malattie amiloidi. Tra queste, ci siamo focalizzati sullo studio dell’alfa-Sinucleina (AS), una proteina coinvolta nella malattia di Parkinson, un disturbo neurodegenerativo caratterizzato dalla degenerazione dei neuroni dopaminergici e l’accumulo di AS in placche amiloidi. Sebbene lo studio delle interazioni AS-dopamina sia di importanza cruciale nella comprensione dei meccanismi responsabili dello sviluppo della malattia, tuttavia, non sono disponibili dati in letteratura riguardo all’affinità di questo legame nei diversi stati ripiegati/funzionali dell’AS. L’elevata tendenza all’aggregazione delle IDP rende difficile il loro studio in soluzione e anche una stima approssimativa dei parametri relativi all’affinità di legame è estremamente difficile. Nuove tecniche di superficie potrebbero pertanto permettere lo studio delle interazioni di legame di molecole e di farmaci capaci di favorire/inibire l’aggregazione. In particolare, le tecniche di superficie potrebbero permettere un migliore controllo dell’immobilizzazione di queste proteine, particolarmente instabili in soluzione, e di studiare con alta precisione fenomeni di legame. Con questa visione, la microscopia di forza atomica (AFM) rappresenta un’opportunità. Il nanografting, una tecnica litografica per mezzo AFM, permette l’immobilizzazione di proteine orientate con il preciso controllo dei parametri di immobilizzazione. Con AFM e AFM-nanografting, abbiamo prodotto una nuova piattaforma per lo studio di diversi aspetti dell’aggregazione/interazione dell’AS. Abbiamo quindi studiato l’affinità di legame tra AS e dopamina misurando variazioni nell’altezza e nella rugosità della topografia AFM su AS immobilizzata in aree confinate, in funzione della concentrazione di dopamina. Il valore micromolare della costante di dissociazione (Kd) è stato inoltre confermato dallo studio dello spiazzamento di un anticorpo legato all’AS in seguito all’aggiunta di dopamina. Sebbene la Kd calcolata con il nostro approccio sia una stima della reale Kd calcolata in soluzione, in quanto influenzata dall’affollamento delle molecole o ai limiti di diffusione, i nostri risultati sono comunque degni di nota. Infine, abbiamo testato il nostro saggio per lo studio delle fasi preliminari dell’aggregazione dell’AS. In particolare, abbiamo osservato come aggregati confinati di AS siano disciolti dall’azione della dopamina, confermando il suo ruolo nell’inibizione della fibrillazione. In questo modo, abbiamo creato un saggio potenzialmente utilizzabile per lo screening di nuove molecole che interagiscono con l’AS ed eventualmente utilizzabili come farmaci. Inoltre, l’interazione di AS con microdomini della membrana cellulare (lipid rafts) sono oggetto di dibattito. Per questo motivo, abbiamo utilizzato dei doppi strati lipidici modello con composizione capace di mimare quella dei lipid rafts. In particolare, abbiamo caratterizzato con AFM e GISAXS (Grazing Incidence Small Angle X-ray Scattering) delle membrane lipidiche di tre componenti, aventi separazione di fase lipidica, studiando l’effetto dell’umidità e della percentuale di colesterolo su ciascuna fase. Abbiamo quindi studiato l’effetto del legame dell’AS su questo sistema modello. In particolare, immagini di topografia AFM in liquido ci hanno dato la possibilità di discriminare la topografia di strutture filamentose formatesi inseguito all’interazione di AS. Il fenomeno, dipendente dalla fase lipidica della membrana e quindi dall’ordine delle molecole, sembra essere favorito da un blando impacchettamento dei lipidi. Inoltre, utilizzando misure GISAXS, è stato possibile determinare un considerevole riordinamento della membrana in seguito al legame di AS.
Intrinsically disordered proteins (IDPs) are natively unfolded, prone to aggregation and directly correlated with amyloid diseases. Among them, we focused on alpha-Synuclein (AS), a protein related to Parkinson’s disease, a neurogenerative disorder characterized by the degeneration of dopaminergic neurons and the accumulation of AS into amyloid plaques. In particular, the study of AS-dopamine (DA) adducts is crucial for the comprehension of the mechanisms responsible of Parkinson’s disease development. However, according to our knowledge, there is no evidence in literature about AS-DA binding parameters at different folding/functional state of AS. IDPs have in fact a strong propensity to aggregate, making it extremely difficult to get even rough estimation of binding affinities in solution. Therefore, new methods able to study surface immobilized IDPs could be useful for both fundamental studies of binding interactions and drug screening of new compounds able to interact favouring/inhibiting the aggregation process. In particular, the use of a surface-based technique could help to better control the immobilization of such proteins, particularly unstable in solution, and to study with high precision binding events. With this perspective, atomic force microscopy (AFM) offers a challenge. Nanografting, an AFM mediated nanolithographic technique, allows for the nanoscale immobilization of proteins in a well-oriented manner with the precise control of the immobilization parameters. By means of AFM and AFM-nanografting, we produced a new platform able to study different aspects of AS aggregation/interactions. First, we studied AS-dopamine binding affinity by measuring the variation of AFM topographic height on AS nanopatches due to binding of DA on the patch as a function of its concentration in solution, and the correspondent surface roughness variation. The value of the dissociation constant, in the micromolar range, was confirmed by studying the displacement of AS-bound mAbs, after the addition of dopamine. We think that this result is noteworthy, even though we are aware that the Kd values obtained with our assay are probably a very rough estimate of the Kd of the AS/DA interaction occurring in solution, due for instance to AS surface crowding and restrictions to diffusion. Finally, the assay was used to study the early stages of the aggregation process. In particular, dopamine dissolved confined AS aggregates confirming its role in the inhibition of fibrillation. In this way, we created a potential screening assay able to study the interaction of AS with new molecules virtually usable as new drugs. Moreover, the interaction of AS with lipid rafts, specialized microdomains of the cell membrane, is object of a strong debate. For this reason, we created a model supported lipid bilayer with composition able to mimic lipid rafts. In particular, we characterized by both AFM and Grazing Incidence Small Angle X-ray Scattering (GISAXS) a three components membrane presenting a lipid phase separation, focusing on the effect of humidity and cholesterol percentage on the structure of each phase. Then, we studied the effect of AS binding on this membrane model system. In particular, trough AFM imaging in liquid we discriminated the topology of filamentous structures resulting from AS binding. The phenomenon is dependent on the membrane’s lipid phase and order and is favoured by mild packing of lipids. From GISAXS measurements, the occurrence of a strong rearrangement of the membrane upon AS binding was suggested.
XXVI Ciclo
1984

Optofluidics is a promising interdisciplinary research and technological field, thanks to its wide potential of application in sector like medicine, chemistry, biology and environmental science. In this context the study of innovative materials including their properties, their efficiency, their limits and their possibility of leading to miniaturized devices is the key point for the overcoming of currently adopted strategies. A promising material that could satisfy new optofluidic requirements is Lithium Niobate (LiNbO3 - LN) thanks to its excellent optical and nonlinear optical properties. In this work we demonstrated for the first time the applicability of the Lithium Niobate as a high intagrable and tailorable substrate for optofluidics. As a matter of fact, we developed all the several stages that can be interconnected to realize a platform with complex optofluidic functionalities: from the droplets generation and manipulation, to their transfer through a microfluidic channel directly engraved on the crystal substrate, to the droplets optical analysis stage. In particular in this thesis we present the first high performant T-Junction droplet generator completely engraved in LN, and the first Ti in-diffused channel waveguide coupled with a microfluidic channel in the same substrate. Furthermore a study on the wetting properties of the Lithium Niobate is discussed. Concerning the optical stage we discuss the realization of optical frequency converter realized in LN, which plays a key role in the development of our optofluidic platform. In fact it can be used to integrate a laser source in the green-blue range that could found application particularly in the biological field. Moreover we present the first frequency converter in the PPLN configuration realized in Zirconium doped LN, a dopants that prevent the optical damage and therefore could increase the intensity of work and the efficiency of conversion of the devices. Also we implemented the process to produce single-mode channel waveguide by Ti in-diffusion as interconnection stage for the optical circuit. Concluding, we were able to implement a well-equipped tool-box for the incorporation of different devices on the same substrate, demonstrating for the first time the integration of all the different stages in a single substrate, and paving the way to an extreme optofluidic integration in Lithium Niobate.
L’optofluidica è un promettente settore di ricerca interdisciplinare con altrettante interessanti applicazioni tecnologiche. Questo grazie al suo ampio potenziale in settori quali la medicina, la chimica, la biologia e le scienze ambientali. In questo contesto uno studio di materiali innovativi che includa le loro proprietà, la loro efficienza, i loro limiti e la loro possibilità di produrre dispositivi miniaturizzati è fondamentale per superare le attuali strategie adottate. Un materiale promettente per soddisfare i requisiti dell’optofluidica è il Niobato di Litio (LN o LiNbO3), un materiale conosciuto per le sue eccellenti proprietà ottiche lineari e non lineari e che qui discutiamo per la prima volta in un contesto optofluidico. In questo lavoro abbiamo dimostrato l’applicabilità del Niobato di Litio come substrato altamente integrabile e adattabile per l’optofluidica. Abbiamo infatti sviluppato tutti i diversi stadi che possono essere interconnessi per realizzare una piattaforma con funzionalità complesse optofluidiche: dalla produzione di gocce, alla loro manipolazione, al loro trasporto in canali microfluidici realizzati nel cristallo, fino all’analisi ottica delle stesse. In particolare nella tesi sono presentati il primo generatore di gocce a giunzione a T completamente fabbricato su Niobato di Litio e la prima guida d’onda a canale in Titanio diffuso accoppiata con un canale. Infine abbiamo proposto il primo studio completo sulla bagnabilità del Niobato di Litio. Per quanto riguarda lo stadio ottico, abbiamo realizzato dei convertitori di frequenza ottica, dispositivi che giocano un importante ruolo nel progetto, in quanto possono essere usati come sorgenti laser integrate con emissione nell’intervallo verde-blu, uno spettro che trova molte applicazioni nell’ambito biologico. In questo contesto abbiamo realizzato il primo convertitore di frequenza con configurazione PPLN realizzato su Niobato di Litio drogato Zirconio, un nuovo tipo di drogante che prevenendo il danno ottico è in grado di aumentare l’intensità di lavoro e l’efficienza di conversione di questi dispositivi. Abbiamo infine implementato il processo per produrre guide ottiche a canale monomodo per diffusione di Titanio, dispositivi necessari per connettere le diverse parti del circuito ottico. In conclusione con questo lavoro abbiamo implementato un’ampia categoria di dispositivi, per la prima volta tutti contemporaneamente integrabili su un singolo substrato. Abbiamo perciò aperto la strada verso un’elevata integrazione di funzionalità optofluidiche su Niobato di Litio.

Parkinson’s disease (PD) current clinical management is mostly based on patient’s subjective report about the effects of treatments and on medical examinations that unfortunately represent only a snapshot of a highly fluctuating clinical condition. This traditional approach requires time, it is biased by patient’s judgment and is often not completely reliable, especially in moderate advanced stages. The main purpose of the EU funded project PD_manager (Horizon 2020, Grant Agreement n° 643706) is to build and evaluate an innovative, mHealth, patient-centric system for PD remote monitoring. After a first phase of research and development, a set of wearable devices has been selected and tested on 20 patients. The raw data recorded have been used to feed algorithms necessary to recognize motor symptoms. In parallel, other applications have been developed to test also the main non-motor symptoms. On a second phase, a case- control randomized multicentric study has been designed and performed to assess the acceptability and utility of the PD_manager system at patients’ home, compared to the current gold standard for home monitoring, represented by symptoms diaries. 136 couples of patients and caregivers have been recruited, and at the end of the trial the system was found to be very well tolerated and easy to use, compared to diaries. The developed System is able to recognize motor and non-motor symptoms, helping healthcare professionals in taking decisions on therapeutic strategies. Moreover, PD_manager could represent a useful tool for patient's self-monitoring and self-care promotion.

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.

The accurate monitoring of cell electrical activity is of fundamental importance for pharmaceutical research and pre-clinical trials that impose to check the cardiotoxicity of all new drugs. Traditional methods for preclinical evaluation of drug cardiotoxicity exploit animal models, which tend to be expensive, low throughput, and exhibit species-specific differences in cardiac physiology (Mercola, Colas and Willems, 2013). Alternative approaches use heterologous expression of cardiac ion channels in non-cardiac cells transfected with genetic material. However, the use of these constructs and the inhibition of specific ionic currents alone is not predictive of cardiotoxicity. Drug toxicity evaluation based on the human ether-à-go-go-related gene (hERG) channel, for example, leads to a high rate of false-positive cardiotoxic compounds, increasing drug attrition at the preclinical stage. Consequently, from 2013, the Comprehensive in Vitro Proarrhythmia Assay (CiPA) initiative focused on experimental methods that identify cardiotoxic drugs and to improve upon prior models that have largely used alterations in the hERG potassium ion channel. The most predictive models for drug cardiotoxicity must recapitulate the complex spatial distribution of the physiologically distinct myocytes of the intact adult human heart. However, intact human heart preparations are inherently too costly, difficult to maintain, and, hence, too low throughput to be implemented early in the drug development pipeline. For these reasons the optimization of methodologies to differentiate human induced Pluripotent Stem Cells (hiPSCs) into cardiomyocytes (CMs) enabled human CMs to be mass-produced in vitro for cardiovascular disease modeling and drug screening (Sharma, Wu and Wu, 2013). These hiPSC-CMs functionally express most of the ion channels and sarcomeric proteins found in adult human CMs and can spontaneously contract. Recent results from the CiPA initiative have confirmed that, if utilized appropriately, the hiPSC-CM platform can serve as a reliable alternative to existing hERG assays for evaluating arrhythmogenic compounds and can sensitively detect the action potential repolarization effects associated with ion channel–blocking drugs (Millard et al., 2018). Data on drug-induced toxicity in hiPSC-CMs have already been successfully collected by using several functional readouts, such as field potential traces using multi-electrode array (MEA) technology (Clements, 2016), action potentials via voltage-sensitive dyes (VSD) (Blinova et al., 2017) and cellular impedance (Scott et al., 2014). Despite still under discussion, scientists reached a consensus on the value of using electrophysiological data from hiPSC-CM for predicting cardiotoxicity and how it’s possible to further optimize hiPSC-CM-based in vitro assays for acute and chronic cardiotoxicity assessment. In line with CiPA, therefore, the use of hiPSC coupled with MEA technology has been selected as promising readout for these kind of experiments. These platforms are used as an experimental model for studying the cardiac Action Potentials (APs) dynamics and for understanding some fundamental principles about the APs propagation and synchronization in healthy heart tissue. MEA technology utilizes recordings from an array of electrodes embedded in the culture surface of a well. When cardiomyocytes are grown on these surfaces, spontaneous action potentials from a cluster of cardiomyocytes, the so called functional syncytium, can be detected as fluctuations in the extracellular field potential (FP). MEA measures the change in FP as the action potential propagates through the cell monolayer relative to the recording electrode, neverthless FP in the MEA do not allows to recapitualte properly the action potential features. It is clear, therefore, that a MEA technology itself is not enough to implement cardiotoxicity assays on hIPSCs-CMs. Under this issue, researchers spread in the world started to think about solutions to achieve a platform able to works both at the same time as a standard MEA and as a patch clamp, allowing the recording of extracellular signals as usual, with the opportunity to switch to intracellular-like signals from the cytosol. This strong interest stimulated the development of methods for intracellular recording of action potentials. Currently, the most promising results are represented by multi-electrode arrays (MEA) decorated with 3D nanostructures that were introduced in pioneering papers (Robinson et al., 2012; Xie et al., 2012), culminating with the recent work from the group of H. Park (Abbott et al., 2017) and of F. De Angelis (Dipalo et al., 2017). In these articles, they show intracellular recordings on electrodes refined with 3D nanopillars after electroporation and laser optoporation from different kind of cells. However, the requirement of 3D nanostructures set strong limitations to the practical spreading of these techniques. Thus, despite pioneering results have been obtained exploiting laser optoporation, these technologies neither been applied to practical cases nor reached the commercial phase. This PhD thesis introduces the concept of meta-electrodes coupled with laser optoporation for high quality intracellular signals from hiPSCs-CM. These signals can be recorded on high-density commercial CMOS-MEAs from 3Brain characterized by thousands of electrode covered by a thin film of porous Platinum without any rework of the devices, 3D nanostructures or circuitry for electroporation7. Subsequently, I attempted to translate these unique features of low invasiveness and reliability to other commercial MEA platforms, in order to develop a new tool for cardiac electrophysiological accurate recordings. The whole thesis is organized in three main sections: a first single chapters that will go deeper in the scientific and technological background, including an explanation of the cell biology of hiPSCs-CM followed by a full overview of MEA technology and devices. Then, I will move on state-of-the-art approaches of intracellular recording, discussing many works from the scientific literature. A second chapter will describe the main objectives of the whole work, and a last chapter with the main results of the activity. A final chapter will resume and recapitulate the conclusion of the work.

Nowadays, nanofluidic platforms are powerful tools for carrying out fundamental studies on molecular-scale phenomena. Typically, the use of these systems results in being crucial both in biomedical and environmental fields. In fact, they are widely exploited for many applications such as detection, concentration, sorting, counting and sizing of several nano-objects such as nanoplastics, viruses, antibodies and DNA. This is the context in which my Ph.D. research project is inserted. I have worked on the development of elastomeric nanofluidic platforms, equipped with different nanostructures, that are the functional areas of the entire fluidic system, useful for different applications among which single nanoparticles detection, high-sensitivity immunoassay analysis and DNA sensing. Typically, nanofluidic platforms are composed of two U-shaped microchannels connected by nanostructures with suitable geometries. All devices were fabricated starting from a pre-patterned silicon mold on which nanostructures were etched using the Focus Ion Beam (FIB) milling technique. Then, the molds were replicated through a, Poly(DiMethylSiloxane)(PDMS) based, double REplica Molding (REM) technique. Although FIB is a high-resolution but expensive technology with REM technique, that is a low-cost and simple approach, I was able to fabricate many polymeric replicas with high precision re-using the mold for several times. This combination allowed obtaining high-resolution nanofluidic platforms reducing fabrication costs, a method that is potentially applicable to processes with high production rate. However, when the dimension shrinks from micro to nanoscale, PDMS presents significant limits. In particular, polymeric nanostructures suffer from the “roof collapse” phenomenon that occurs when the replica is sealed with a glass substrate, a necessary procedure to obtain watertight devices. It is possible to overcome this problem both by exploiting the Junction Gap Breakdown (JGB) technique and by using hard-PDMS (h-PDMS) during the fabrication process. During my Ph.D. research activity, I have initially worked on an asymmetric structure that was a funnel-shaped nanochannel in which the tip, after experiencing “roof-collapse”, was re-opened, thanks to the Junction Gap Breakdown procedure. From an electrical investigation of the devices fabricated with this strategy, we observed an ion current rectification characteristic and analyzing the electro-kinetic transport properties we observed that, in few minutes, intra-funnel accumulation occurs, and this phenomenon results in being stronger for low ionic strength solutions. Combining intra-funnel accumulation of biomolecules, governed by electro-hydrokinetic phenomena, that occurs applying high voltage across the device, and an appropriate functionalization of nanochannel polymeric surface with antibodies, it was possible to decrease sensing limit for the detection of one or several targeted antigens for clinical diagnostics. It was possible to identify through fluorescence optical microscopy and electrical measurements, the uptake of a specific antigen, diluted in solution (down to 1 pg/ml), to the nanochannel surface when functionalized with antibodies. So, in this condition, we successfully detected antigen-antibody binding on the nanostructure surface, a promising step for realizing a high-sensitivity nanofluidic immuno-assay sensor. Successively, I have developed other nanofluidic devices equipped with symmetric nanostructures for single-particle sensing. These devices were made using h-PDMS (hard- PDMS) in order to confer higher rigidity to the nanostructures, i.e. the functional part of the device, avoiding collapse problems. H-PDMS was used in exploiting a “focused drop-casting” approach in order to make only the nanostructure region stiffer, while leaving the other regions of the device flexible enough to avoid the formation of cracks along the device. Combining the nanoscale dimension of the sensing gate with the Resistive Pulse Sensing (RPS) technique, it was possible to analyze single nanoparticles (NPs) and the motion of single λ-DNA molecules through the nanochannel as transient variations in ionic current during the translocation events, allowing a real-time, label-free and high-sensitivity detection. In particular, it was possible to demonstrate the possibility of counting nano-objects depending on selected characteristics (i.e. charge and size ranging from 40 nm to 100 nm) that is a crucial step, useful in many fields such as medicine (drug delivery, imaging, cell-secreted carriers), environment (groundwater remediation, nanoplastics detection) and food production (nano-agrochemicals, nano-encapsulated additives, anti-microbials).

Fused Filament Fabrication (FFF) three-dimensional printing have attracted much attention for fabrication of microfluidic platforms used to construct electrochemical microfluidic biosensors because of high process speed, low production costs and the possibility of manufacturing directly from virtual data. Because of poor adhesion between metal electrodes fabricated using conventional techniques and FFF printed thermoplastic substrates, electrodes are usually integrated into the devices either modularly or using adhesive layers placed at the bottom of fluidic channels. These have hindered the exploitation of FFF for scale-up manufacturing of monolithically integrated microfluidic biosensors. In this work, supersonic cluster beam deposition (SCBD) was employed to fabricate strongly anchored nanostructured electrodes integrated into FFF printed microfluidics platforms. SCBD enables the formation of well-adhering metallic thin film electrodes by implanting supersonically accelerated neutral metal clusters into polymeric substrates. The SCBD also enables deposition over large areas using noble metals and metal oxides with precisely controlled geometry and surface topography. A novel integrated manufacturing approach was developed and optimized to couple SCBD fabricated electrodes with consumer-grade FFF printed microfluidics, employing acrylonitrile butadiene styrene as the base material, to develop a three electrodes configuration electrochemical sensor on-a-chip. Electrochemical investigation performed using stagnant ferro/ferricyanide probe showed that the integrated device possesses high sensitivity and functionality as an electrochemical sensor. In addition, in-channel laminar flow electrochemical detection conducted using the same probe showed robust stability in the system response for online dynamic detection. The integrated platform could be employed for various customized clinical, industrial, and environmental sensing applications.

Since the growing interest in Lab-On-a-Chip (LOC) applications, the demand for a high multifunctional and portable devices in order to increase the achievement of complex laboratories analysis on a small size chips. This need pushed the research to integrate in the substrates several tools with different tasks, and consequently, the requirements of the material exploited become rapidly more restricted. In particular, the performances of multiple toolkit device are related to its capabilities of combining and matching stages, thus avoiding any detrimental interferences between the material properties needed for their realization. During the last 20 years, several materials have been proposed for the combination of different tools on the same device. Only recently our group proposed Lithium Niobate (LiNbO3) as a valid alternative for a monolithic substrate in LOC application. This material is well-known in the field of integrated optics, due to its interesting optical properties, that brought it to be the main component in optical modulators in the telecom devices. The excellent performances in this field can be combined to the microfluidic for the realization of reliable optofluidic stages for LOC application. Moreover, LiNbO3 was demonstrated as a substrate for several micro-manipulation tools, exploiting its further properties, such as photovoltaic tweezers of micro-sized objects, nano-droplets pipetting with pyroelectric induced field, acoustic driven particle transports via piezoelectric generated surface acoustic wave respectively. For these reasons, the merge of all these tools on the same monolithic substrate could represent the optimal improvement to push even further the LOC technology. This thesis aimed to demonstrate that a multifunctional opto-microfluidic platform can be realized in one monolithic substrate of lithium niobate, achieving properties that can be properly exploited for LOC application. In particular, the attention was focused on combining integrated optics and microfluidics in a unique platform, where light is confined in an optical waveguide and crossed a microfluidic channel allowing for a transmission detection. The realization of microchannel for the coupling with microphotonic structures is presented: so in particular channel configurations, such as droplet generator, are characterized and compared to those achieved with other standard material, in order to show the better performances provided by LiNbO3. In this platform the crucial aspect was played by the optical quality of the lateral walls of the microfluidic channel in order to guarantee an optimized optical coupling of the integrated optical waveguide and the microchannel. Such an optical grade quality allows for the integration of microchannels with Ti-indiffused waveguide, in an unexplored way alternative to the standards in optofluidics. This optofluidic coupling is performed by crossing the waveguide with an engraved microfluidic channel. This configuration enables the analysis of the transmitted light guided by the first half of the waveguide to the second half of it. The self-aligned geometry allows for the traveling of the light beam from one waveguide (input waveguide) across the channel until it recouples in the other part of the waveguide (output waveguide). This optical transmission signal of the medium inside the channel is exploited for LOC applications, such as optical measurements of the droplet geometrical properties (such as frequency, length, volume), and controlled microfluidic pH titration, which allows for the pH determination and the neutralization of strong acidic or basic solution, and finally as an optical multiplexer actuated microfluidically. In all these applications, the devices showed not only high level of integration and accurate response, but also superior performances than the standard procedures and materials. Moreover, a further tool was integrated to this optofluidic platform: a photovoltaic tweezers, exploiting the lithium niobate capability of photoinducing electric field on its surfaces. These multifunctional stages are proposed for the effectively manipulation of the orientation liquid crystal inside a microchannel, and the consequent polarization control waveguide transmission beam.

The framework of my thesis are Biomedical (or Biological) Microelectromechanical Systems (BioMEMSs). Two fields in which this discipline is involved are sensors and fluidics. Functionalized organic materials are under investigation to be the means for target biological sensing, and sensors are evolving to be integrated in fluidics platforms in order to produce in the future new small portable diagnostic devices. On the other hand one of the challenges of micro and nanofluidic technology is the fabrication of drug release devices, in order to control the amount of drug present in an organism. In this thesis these two arguments are considered. First we will discuss the implementation of a process oriented to the fabrication of an hybrid Organic Field Effect Transistor (OFET) with sensing capabilities from the semiconductive layer. In the second part we will show the fabrication process of a silicon based structure for the scaled-up characterization of drugs in nanochannels for controlled drug release. The characterization will consider charged microspheres playing the role of drugs to be tracked with a microscope. We will highlight also the possibility of implementing the transistor related technology in nanofluidic systems for the electronic controlled drug release.

The focus of this Ph.D. thesis is the investigation of the opto-electronic performances and photonic characteristics of Organic Light Emitting Transistors (OLETs). These promising multifunctional devices unify the switching properties of transistors with the light emission capability of light-emitting diodes. The OLETs that have been studied in this research present a peculiar trilayer configuration, which can allow to obtain high light emission efficiency by reducing the quenching processes inherent to the device structure. For these devices, the exciton formation, the light outcoupling and the mechanisms responsible for light losses were investigated, aiming at fully disclosing the potentiality of OLETs in therm of External Quantum Efficiency (EQE) and brightness. In addition, the possibility to modulate the width of the emission area by the gate voltage up to extension of the entire channel was demonstrated. This result is of unprecedented importance for allowing the implementation of OLETs in lighting and photonic applications. Furthermore, a novel strategy based on the introduction of a non conventional planar photonic structure into the device architecture was used to increment the light emission efficiency. Indeed, a fully-organic multilayer structure, that worked both as Distributed Bragg Reflector (DBR) and as gate dielectric, was iserted into the OLET architecture to turn the gate dielectric into an optically active component, capable of enhancing light extraction in forward direction by reducing total internal reflections processes. Other unidimensional photonic structures for lasing application were designed, fabricated and characterized. In specific, a linear Distributed Feedback structure (DFB) based on silk was used for realizing a biocompatible, biodegradable and edible organic laser. The entire route for extracting, purifying and manufacturing silk was optimized for achieving the best performing photonic component. Moreover, a method for making optically active silk by feeding directly the larvae with lasing dye molecules was introduced; the method is green because it allows to fabricate intrinsically colored silk by eliminating the need of resources as water, energy and organic solvents. These findings may open perspectives for applications of optically active silk in biophotonics and biological sensors, such as the realization of Lab-on-a- Chip devices for the biodiagnostics.

In recent years it has been shown that the outstanding properties of graphene, a direct consequence of its unique 2D structure, could be further tailored by surface functionalization with suitable materials, towards a fine tuning of the system's physical and chemical properties. In particular, the covalent functionalization of graphene using organic functional groups has been explored as a pivotal step towards the formation of graphene composites at the nanoscale. Alongside the commonly diffused approach with diazonium salts (abundant and quick but hard to control), a more selective and controlled method has been shown as very promising: 1,3-dipolar cycloaddition (1,3-DC) of azomethine ylide has been investigated for the chemical modification of graphene-like systems. However, while graphene's high specific surface area of 2630 m^2/g provides numerous possible binding sites, its chemical inertness makes it difficult to modify graphene's structure without disrupting it or introducing excessive disorder. Thus, to finely control or intentionally design the binding sites of functionalizing molecules on the surface of graphene while preserving the high quality of its unique structure remains an open challenge. A promising route in order to locally improve the reactivity of graphene is to introduce beneficial structural defects. For example, due to the defect-induced electron charge redistribution, defective graphene shows increased chemical reactivity towards addition reactions. At the same time, the precise control in defect formation would allow a fine tailoring of the surface chemistry of graphene, fundamental for the engineering of its electronic properties or for sensing applications. The most versatile approach that satisfies the requirements for a controlled introduction of structural defects in graphene is based on particle irradiation techniques. Indeed, effective defect modulations can be patterned over a large area via electron beam irradiation (EBI), utilizing scanning electron microscopy (SEM), in a very flexible way. The PhD research presented here builds upon this idea. Covalent functionalization of different graphene-based systems has been achieved, allowing to explore various parameters of the functionalization process, including EBI defect-engineering. Firstly, the functionalization procedure is optimized utilizing graphene nanosheets (GNS) and reduced graphene oxide (rGO) dispersed in the liquid phase, and, for the first time, a comparison of the efficiency of 1,3-DC of azomethine ylide in different dispersant solvents (NMP and DMF) is reported. The functionalization is confirmed with electron energy loss spectroscopy (EELS) and energy-dispersive X-ray spectroscopy (EDX) measurements, and new Raman features arising from the functionalization with azomethine ylide are detected. Density functional theory (DFT) models for pristine and functionalized rGO are built and characterized by evaluating the restrained electrostatic potential (RESP)-derived partial atomic charges, which highlighted the localization of the charges in the pristine rGO induced by the presence of defects (epoxy groups) in the initial structure. Furthermore, the computation of the power spectrum (PS) helps with the assignment of characteristic Raman peaks to the functionalization with the azomethine ylide. Finally, the elemental composition of pristine and functionalized graphene is investigated via x--ray photoelectron spectroscopy (XPS) measurements, allowing to confirm the stability of the functionalization (up to 180 °C) and to estimate the efficiency of the 1,3-DC on graphene. Thanks to the local inhomogeneity of the partial charges, due to the presence of oxygen functional groups in the initial structure, a higher functionalization is achieved on rGO (~ 2 times higher than on GNS). The enhancement of the chemical reactivity measured in our defected graphene validates the interest in further exploring the possibility to control the position of defects on higher quality graphene systems. Defect patterns are designed on micromechanically exfoliated graphene flakes on silica substrates by EBI. Their distribution is analyzed with Raman spectroscopy, revealing that surface treatments of the graphene-supporting substrate have strong impact on the lateral resolution that can be achieved on the final defective pattern. Unintentional defects-rich zones are revealed in the adjacent parts of the irradiated areas, and Monte Carlo simulations of primary electrons scattering events demonstrate that these transition zones originate within the area where back-scattered electrons (BSEs) and secondary electrons (SEs) generated near the substrate surface by BSEs (by interaction with organic impurities adsorbed on the Si/SiO_2 substrate) escape from the substrate surface. These results can be exploited in order to design high spatially-resolved defect patterns on monolayer graphene flakes, introducing a selectively enhanced chemical reactivity towards the organic functionalization. To explore this route, defect patterns are designed on exfoliated graphene flakes via low-energy (30 keV) EBI. Raman spectroscopy maps show the appearance of the characteristic D peak only in the patterned area, while AFM images confirm the spatial resolution of the pattern (~ 100 nm). The 1,3-DC of azomethine ylide in-situ involves the localization of a C=C bond of the graphene structure, which is favorable in presence of the defects, hence introducing a selective control of the chemical modification of graphene. The Raman analysis on functionalized graphene flakes exhibits new features only in the patterned area, while the unexposed area still presents the spectrum of pristine graphene, confirming the selectivity introduced via defect patterning. Moreover, AFM images of patterned graphene show an improved adhesion on the silica substrate, allowing to avoid detachment issues during the functionalization procedure in the organic solvent. DFT allows to identify the vibrational contributions of the functional groups of the azomethine ylide grafted on the graphene surface and of the modified vibrational modes of the graphene lattice in the experimental Raman spectrum. Furthermore, under laser irradiation (up to 1.6 mW) the Raman spectrum recovers towards the spectrum of non-functionalized patterned graphene, indicating the desorption of the ylide and the reversibility of the functionalization. Then, the functionalization of epitaxial graphene (EG) on SiC is investigated, benefiting from the valuable addition of scanning tunneling microscopy (STM) and spectroscopy (STS). STM images of functionalized EG reveal the appearance of new structures, randomly arranged over the flat terraces (with lower density) and along the edges (with higher density), with an average height in the range 2 - 15 A, and a graphene surface coverage of ~ 14 %. The graphene structure is preserved after the functionalization procedure, as confirmed by atomically resolved STM images of its hexagonal lattice. STS spectra acquired on functionalization EG indicate the opening of a bandgap (of 0.13 - 0.20 eV) in the local density of states (LDOS) of these structures, in contrast with the zero-gap linear behavior measured on graphene. The Raman analysis of functionalized EG exhibit new features, together with a downshift of the G and 2D peaks. These results indicate the grafting of azomethine ylides on graphene. Finally, to increase the efficiency of the covalent functionalization of EG and, in particular, to be able to spatially design the functionalization of EG, defect patterning via EBI is explored. After patterning, Peak Force - Quantitative NanoMechanical (PF-QNM) measurements allow to identify the designed defect pattern, confirming the spatial resolution of the technique (with different electron doses and e-beam scan step sizes). Moreover, the analysis of the adhesion forces reveals that the patterning results in an enhancement of the adhesion of the graphene with the substrate, as already seen in previous experiments. Although incomplete, these are valuable results in the outlook of a deterministic and controlled chemical functionalization of EG on SiC, which would be extremely beneficial for the fabrication of high quality devices at the nanoscale. In fact, EG on SiC eliminates the need for transfer procedures and presents favorable characteristics for large-scale graphene electronics. The results discussed here open the route for a controlled functionalization of different graphene-based systems with designed molecules, which could act both as active functional groups or passive spacers towards multi-functional sensing devices or multilayered spaced graphene systems optimized for hydrogen storage or gas sensing.

The control of cell growth, proliferation and differentiation on biomaterials surfaces is of fundamental importance for regenerative medicine, prosthetics, or cell-based assays. The microfabrication of cell-on-chip platforms based on a new family of poly (amidoamine) hydrogels, are promising for in vitro and in vivo applications. Hydrogels present characteristics that mimics biological environments, such as the cross-linked nature of the extracellular matrix, the tissue properties (high water content), and the permeability to oxygen and metabolites. Hydrogels based on poly (amidoamine) results in an optically transparent, biocompatible and fully biodegradable substrate recommended for body implants that are minimally invasive, and naturally eliminated by human body. In my PhD work I intended to use microfabricated hydrogels for fine-tuning the contact guidance of cells. As microfabrication tools I set up reaction injection moulding for producing features down to 100 µm and developed a novel approach relying on electron beam lithography. This innovating microfabrication consists in the ability of directly writing patterns on already cross-linked hydrogels, with the capability of producing structures at sub-micrometric scale. The exposure to the electron beam produces particular modifications enabling the control of physico-chemical properties of irradiated area. I obtained a selective attachment of proteins as a function of the electron-beam dose; an exclusive adhesion and growth of neural cells on the exposed surfaces; and the control of neurite outgrowth guidance along a microfabricated network. These results offer new perspectives to build physiological microenvironments or cell-on-chip platforms, based on a novel class of microfabricated hydrogels.

Photoactive semiconductors are a hot topic of research due to their applications in environmental remediation, photovoltaics, smart devices, light-activated synthesis and self-cleaning surfaces. Among them, oxide semiconductors play a main role thanks to their wide availability, stability, ease of preparation and tunable surface properties. In this context, my Ph.D. focused on the application of oxide semiconductors for three main purposes: pollutant remediation, photocatalytic synthesis of hybrid materials, and smart systems for the controlled release of active substances. Alongside this main research project, I developed two original hands-on activities based on oxide systems for public engagement. Oxide semiconductors for environmental remediation. Photocatalysis is an advanced oxidation process that can achieve the complete degradation of contaminants without the addition of reagents. However, its real-life application has been hindered by several limitations, such as the use of nanosized powder, costs of light irradiation, possible accumulation of toxic reaction intermediates and the sensitivity to complex water matrices. During my Ph.D., I investigated the deposition of photocatalyst powder on macroscopic devices based on aluminum plates for air purification. Aluminum is a cheap and technologically-relevant substrate, but its application as substrate for photocatalyst immobilization has been hampered by adhesion issues and metal ion diffusion within the photocatalytic layer that increases recombination of photogenerated carriers. Thus, I investigated the use of silica interlayers to promote adhesion, efficiency and reusability of TiO2 films on aluminum plates. Films were prepared from stable titania sols and deposited on aluminum substrates with different surface morphology and with silica interlayers of different thickness. The study of the coating structure, morphology, optical properties, adhesion and hardness showed that the nature of the substrate and its surface roughness determined the optimal number of silica interlayers. When the silica interlayer was too thin, moderate cracking was still observed, whereas a too thick silica interlayer led to peeling off of the film. The use of rougher surfaces, as in the case of sand-papered aluminum, required a higher number of silica layers to promote a more homogeneous surface where the titania coating could effectively adhere. However, the addition of a thicker silica layer did not erase the effect of the sand-paper pre-treatment on surface roughness. Films on sand-papered substrates showed promoted photocatalytic activity with respect to the smoother counterparts, possibly due to their larger exposed contact area. The prepared films exhibited excellent light-induced superhydrophilicity and self-cleaning properties towards fouling agents (alkylsilanes). Photocatalytic degradation tests were carried out using both a model volatile organic compound (ethanol) and NOx. The silica interlayer proved crucial to promote the film robustness, effectively increasing the mechanical stability and reusability when a thicker interlayer was adopted on sand-papered aluminum plates. In order to cut the costs associated with lamp irradiation, the visible-light promotion of large band gap photocatalysts is a widely investigated approach. In this regard, I studied the modification of TiO2 with Sn and N species aiming to improve the photocatalyst visible-light absorption for the solar-light photocatalityc degradation of emerging pollutants. Three different synthetic routes were investigated: a bulk synthesis, where Ti and Sn precursors were both added in the sol-gel synthesis, a seeded procedure, where pre-formed SnO2 crystals were added to TiO2 synthesis, and a mechanical mixture, where the oxides were mixed together then calcined. Marked differences were observed in the final composites’ structural, morphological and optical properties, leading to notable changes in the photocatalytic performance. Interestingly, bulk and seeded samples showed notable photochromic properties under UV light, which varied based on the doping level: this is the first time photochromic effects have been observed in Sn-promoted TiO2. These findings can be related to the different nature of the defects introduced in the oxide lattices depending on the synthetic route, which reflect in the photocatalytic performances of the modified semiconductors. The photocatalytic degradation of wastewater pollutants in complex matrices requires a close scrutiny of the generated byproducts to avoid possible accumulation of intermediates even more toxic than the parent compound. In this respect, I determined that the degradation of tetracycline, a widely used antibiotic, by a benchmark TiO2 sample, despite the fast pollutant disappearance, leads to poor mineralization and byproduct accumulation, especially in the presence of common electrolytes, such as HCO3-. Conversely, the use of commercial ZnO samples with the same surface area resulted in a faster tetracycline degradation kinetics and a much higher mineralization degree compared to TiO2 in all the investigated water matrices. These results can be attributed to different photo-degradation pathways followed by the two oxides, as shown by tests with radical scavengers and by-product analyses. While TiO2 degradation pathways are strongly dependent on both hydroxyl radicals and holes, ZnO mineralization activity is mostly related to holes, which limits the interference of •OH-scavenger species such as bicarbonates. Photo-induced synthesis of oxide-polyaniline composites for environmental remediation. To date, photocatalysis remains a comparatively slower and costlier wastewater treatment compared to adsorption. For this reason, during my Ph.D I also investigated new generation adsorbents characterized by easier regeneration and ability to perform a controlled release of the adsorbed species to be further treated or reused. To this aim, I investigated polyaniline (PANI) composites prepared via an innovative photocatalytically-induced synthesis. PANI materials have been recently adopted as sorbents for environmental remediation due to their stability, redox properties and acid-base characteristics. However, PANI traditional oxidative synthesis (here labeled as PANI-aniline) adopts noxious and toxic reagents (aniline and (NH4)2S2O8) and leads to carcinogenic by-products and large amounts of waste. The alternative photocatalytic approach I developed is a two-step synthesis starting from aniline dimer (N-(4-aminophenyl)aniline) and exploiting TiO2 photocatalyst to initiate the oligomerization, and a greener oxidant (H2O2) in the polymerization step. The resulting PANI-TiO2 nanocomposites showed very different structural, morphological and surface properties with respect to PANI-aniline, resulting in fast and efficient removal of water pollutants. To better understand the reaction pathway and tailor the material properties, the relative roles played by TiO2 and H2O2 in the synthetic procedure were investigated in depth. UV-irradiated TiO2 was found to promote PANI crystallinity and polymer-oxide interactions. The amount of added H2O2 has a crucial role on the composite properties by promoting either surface growth of PANI chains or polymerization in the liquid bulk. High H2O2 amounts seem to promote a homogenous polymer formation mechanism, leading to nanocomposites with high PANI content and thermal stability, but low crystallinity degree and surface area. Low H2O2 quantities give rise to highly porous, large surface area nanocomposites with good crystallinity but low PANI content. The latter samples exhibited the best performance in pollutant sorption tests, achieving a fast and complete removal of dyes and heavy metals also in the presence of electrolytes. These samples also showed reusability in consecutive stress tests and could be regenerated simply by treatment with alkaline aqueous solution at room temperature. The next step was to investigate the role of the nature and morphological features of the semiconductor: commercial TiO2 photocatalysts with either 50 m2g-1 (labeled TiO2-P25) and 12 m2g-1 (TiO2-Kronos) were compared with WO3 either lab-synthesized (3.5 m2g-1, named WO3-Synt) or commercial (6.1 m2g-1, WO3-Comm). The composites showed a nanorod / nano-wire morphology: the length of the polymeric rods and the embedding of the oxide particles within the polymer network strongly depended on the nature and morphology of the photocatalyst. Furthermore, while > 80% total dye removal capacity was observed for all samples (with the exception of PANI-WO3-Comm), notable differences were observed in terms of released tests. In particular, PANI-oxide composites consistently showed dye-release capacities far higher than PANI-aniline. The ease of desorption opened the door to the facile regeneration of the adsorbent and to the adsorbate recovery for its recycle in a circular economy perspective. Therefore, I investigated an adsorption-photocatalysis coupled system which exploited the reversibility of the pollutant removal process. In particular, after consecutive dye adsorption cycles, the contaminant was released by the PANI-oxide adsorbent and subsequently mineralized by a ZnO driven photocatalytic process. The nature of the adsorption process was deeply investigated and selectivity tests with cationic and anionic dye mixtures proved the preferential adsorption of PANI-oxide adsorbents towards anionic dyes. In the end, the promising and reversible adsorption capability of PANI composites prompted me to investigate their possible application in CO2 capture systems. Thus I have worked on reviewing the literature works on the topic, comparing the performances of different PANI materials towards CO2 removal. Smart systems based on light-responsive oxides. The intrinsic characteristics of semiconductor oxides, such as their photocatalytic and surface properties, can be exploited in the design of smart systems for the controlled release of unstable active substances, such as essential oils. Among them, cinnamaldehyde (CIN) is a low-cost natural compound endowed with antibacterial, anti-cancer, antifungal, and anti-inflammatory properties. However, CIN has poor water solubility, high volatility and very poor stability in environmental conditions, undergoing degradation when exposed to heat, light or even oxygen. These issues hinder CIN applicability, thus smart systems able to store this active substance and to safely release it at will, are of extreme interest for the scientific community. In this context, during my Ph.D. I developed oxide-based hybrid systems for the release of CIN catalyzed by acidic pH. The smart system was obtained by a grafting method based on amino-silane linkers and imine chemistry: (3-aminopropyl)triethoxysilane (APTES) was adopted for the functionalization of the oxide surface. The terminal amine group of the silane (-NH2) was used for a condensation reaction with the aldehydic group of CIN (-HC=O), yielding an imine bond (-HC=N-) between APTES and CIN and a loading of ca. 5 molecules/nm2, determined with CHN and TG analyses. The covalent grafting of cinnamaldehyde, showed by FTIR spectra, preserved the molecule stability, simplifying storage. Release tests were performed at pH values between 5.0 and 7.4: thanks to the pH-sensitivity of imine bonds, a fast CIN release was observed at pH 5.0. The grafting procedure was also performed on a porous semiconductor film, demonstrating the versatility of this method. Exploiting the oxide photoactivity, the fouled film was regenerated upon 1h UV irradiation, opening the door to reusable devices for CIN controlled release. Besides the conventional approach of loading bioactive compounds on solid drug carriers, smart systems based on particle-stabilized emulsions (i.e., Pickering emulsions) are receiving increasing attention from the scientific community. In this regard, during my last year of Ph.D. I investigated oil-in-water Pickering emulsions prepared with food-grade vegetable oils and stabilized with bare ZnO particles. FTIR studies highlighted that, during emulsification, ZnO particles undergo an in situ functionalization by fatty acids present in the vegetable oil. This procedure gives rise to very stable and homogeneous emulsions (mean droplet size ca. 1 μm). Confocal microscopy images demonstrated the high stability of the system towards long time storage (more than 9 months), temperature variations, mechanical stress and increased ionic strength. ZnO-Pickering emulsions were loaded with CIN in the oil phase, in order to store the active molecule and release it at will by the application of five different stimuli. In particular, thanks to the semiconductor and amphoteric properties of ZnO, the developed smart system was able to release CIN by switching to a water-in-oil Pickering emulsion when subjected to acidification, UV and solar light irradiation, CO2 bubbling and the addition of bi/trivalent cations. This is the first report of an emulsion system responsive to five different stimuli. Depending on the type of stimulus, either a burst release or a controlled release over the course of several hours could be achieved. The emulsion switching can be attributed to the oxide surface charge: when ZnO is negatively or slightly positively charged, the oil-in-water emulsion is stable, while, when the oxide surface has high positive charge, the oil droplets’ intrinsic negative charge is neutralized and coalescence phenomena occur. A more positive ZnO surface charge can be achieved through the addition of acidic species (such as H+ and H2CO3 via CO2 bubbling), multivalent cations, which give specific adsorption on ZnO surface, and through light irradiation, which activates the photocatalyst and generates acidic species. The starting oil-in-water emulsion could be reobtained by basification, N2 bubbling and storage in the dark. The ZnO Pickering emulsions were able to safely store and release CIN molecules, which did not undergo any degradation neither during storage, nor after release in water solution. In the end, I have contributed to a work on near infrared (NIR)-emitting GdVO4:Nd systems. This composite material proved promising for bioimaging applications, thus, I exploited my experience oxide synthesis to investigate the role of the synthetic procedure on the material properties and NIR-emitting activity. Moreover, test on GdVO4:Nd functionalization with silane molecules (octylsilane and APTES) were carried out. The modification of the material surface with organic compounds can led to a possible increase in the material biocompatibility, as well as to the possible grafting of active molecules, such as cinnamaldehyde, for application in theragnostic. Chemistry dissemination activities. During my PhD, I was involved in chemistry dissemination activities in the framework of the “Piano Lauree Scientifiche, PLS”. In this context, I helped to develop two laboratory activities for high school teachers and students. The first one, aimed at teaching the basic concepts of surface science, focused on the preparation of superhydrophobic coatings based on films of surface functionalized oxide particles. The film’s superhydrophobicity was tested for different applications (anti-stain, self-cleaning, liquid transportation) and compared with model hydrophobic, hydrophilic, and superhydrophilic surfaces. The second activity mimicked the chemistry of stained glass, introducing basic concepts of redox reactions, chemistry of color, and plasmonic nanoparticles. Stained glass colors were copied through the deposition, on glass slides, of silica coatings colored by metal ions and nanoparticles. A silica sol was used as matrix to embed metal ions, which were reduced in situ by thermal treatment on a hot plate. The formation of metal nanoparticles by this procedure induces plasmonic colors in the glass coating, thus “mimicking” the ancient procedure of stained-glass fabrication. These works led to two publications on the Journal of Chemical Education.

Current photovoltaic (PV) market is strongly dominated by an intense use of silicon. Although it is the second most abundant element on the Earth crust, after oxygen, Si is never present in its pure form but always bounded with other elements, and relatively complex and expensive purification procedures are needed in order to have clean, crystalline and optimally doped pure silicon. This issue, joined with the ever-increasing demand of clean Si by almost all the technological modern applications, led scientists all over the world to look for suitable alternatives. One of the most promising options, is to try to substitute silicon with carbon, essentially for two reasons: (i) pure C not only exists in nature but can also be obtained and purified through easy and low-cost processes, (ii) carbon can behave as a metal or a semiconductor without being doped, depending only on the particular allotrope. Moreover, carbon allotropes capability of arranging in various geometry allows C-based materials to assume different dimensionality, starting from the quasi zero-dimensional fullerene to three-dimensional diamonds. This makes carbon nanomaterials excellent candidate for a wide range of electrical and technological devices, offering the possibility to chose the suitable allotropes depending on the particular task that is needed to be fulfilled. For photovoltaic application, a semiconducting material which can provide dissociation sites for excitons is necessary. To accomplish this role, the mono-dimensional form of C, carbon nanotubes (CNTs), revealed to be a perfect substitute of p-type silicon, on one side of the junction because CNTs are naturally p-doped in air. Moreover, thanks to their peculiar geometry and extraordinary electrical conductivity, they are able to provide excellent transport path for the dissociated carriers with a very good transparency (which allows a relevant amount of incident light to reach the depletion region). In the first chapter of this thesis, carbon nanotubes will be introduced, emphasizing the properties which make this nanostructured materials optimal for PV applications. Then, the different types of carbon/silicon heterojunctions will be analyzed, starting from the classical semiconductor theory, to a more complex and realistic model. At the end of the chapter CNTs solar cells state of the art will be presented, highlighting the open questions at which this thesis is aimed to answer. The experimental techniques, such as angle-resolved X-rays photoelectron spectroscopy (AR-XPS) and transient reflectivity (TR) measurements, used to reach this goal will be presented in Chapter 2, together with the description of the manufacturing processes that yielded to the creation of three different series of PV devices, with an improvement of the efficiency from 0.1% to 12.2% in three years. In the third chapter, we will show how the complex buried interface between CNTs and Si can be investigated and modelled by means of photoelectron spectroscopy techniques. A complex oxide interface, composed by silicon dioxide and non-stoichiometric silicon oxide, has been unveiled and possible effects on the power conversion efficiency of PV devices are outlined. A systematic study on the chemical and physical properties of the buried interface will be presented in Chapter 4. Oxides have been alternatively removed and regrown using suitable acids and the effects on the PV performances will be discussed in detail in this chapter. The doping effects of acids on the carbon nanotubes will also be investigated through Raman spectroscopy. Acid effects on the heterojunctions will be unambiguously shown by the XPS measurements, and the matching of these data with the electrical PV measurements allows us to discuss the nature of the heterojunction in more detail. In order to properly address the operation mechanism of these devices, which can be either a conventional p-n or a metal-insulator-semiconductor (MIS) junction, the dynamics of charge transfer processes at the interface will be investigated in Chapter 5 with time-resolved pump-probe reflectivity measurement. The aim is to find a correlation between the thickness of the buried SiOxlayer and the carriers photogeneration and transport, comparing the device electrical parameter with the ultrafast behavior, analyzed by time-resolved reflectivity. These last findings, along with several improvements in the CNTs dispersion and deposition, have led to the creation of optimized third-series solar cells with a record efficiency of 12.2%, which will be fully characterized at the end of this last chapter through a combination of suitable experimental techniques, in order to highlight the factors which contributed to this huge jump in the power conversion efficiency. The stability in time of this optimized PV devices will finally be discussed.

In recent decades, surface plasmon resonance has known a growing interest in the realization of miniaturized devices for label-free sensing applications due to the need of increasing the sensitivity of the sensor and limiting the consumption of material. This work is aimed to the realization of plasmonic nanostructures that can be applied to different sensoristic fields in order to create a starting point for the realization of miniaturized sensors for a wide range of applications. In this context a careful study of geometry and materials suitable for creating the starting plasmonic platforms (i.e. gold sinusoidal gratings) was performed and a characterization method has been optimized. Subsequently a manufacturing strategy that would allow to obtain a large number of versatile substrates in a short time and in a cheap way was designed. Thus by combining interference lithography and soft lithography the required plasmonic substrates were realized and characterized by varying the azimuthal rotation of the grating. The substrates were tested in different application fields: the detection of M. tuberculosis DNA using PNA probes, the detection of cystic fibrosis DNA using DNA probes, the detection of explosive trace and the detection of L. pneumophila bacteria. The reached optimization and control of the sensing experiment and plasmonic surface preparation procedures, and the obtained results, have shown the extreme versatility of the sensors realized with respect to different applications. This goal is to be considered a good starting point for future studies aimed to the miniaturization and engineering of a sensor suitable for different needs and applications spacing from the biomedical field to the security one, passing through food and pollution analysis.
Negli ultimi decenni la risonanza plasmonica di superficie ha conosciuto un crescente interesse nella realizzazione di dispositivi miniaturizzati per applicazioni sensoristiche label-free dettate dalla necessità di aumentare la sensibilità dei sensori e di limitare il consumo di materiale. Questo lavoro ha come scopo la realizzazione di nanostrutture plasmoniche che possano essere applicate a diversi campi della sensoristica in modo da creare un punto di partenza per la realizzazione di sensori miniaturizzati per molteplici applicazioni. In primo luogo è stato effettuato uno studio accurato della geometria e dei materiali adatti alla realizzare delle nanostrutture (grating sinusoidali metallici nella fattispecie) ed è stato ottimizzato il metodo di caratterizzazione delle superfici plasmoniche. Successivamente è stata ideata una strategia di fabbricazione che permettesse di ottenere un grande numero di substrati versatili, in poco tempo e con costi limitati. Così combinando litografia interferenziale e soft lithography sono stati realizzati dei substrati plasmonici caratterizzati variando la rotazione azimutale del grating. I substrati sono stati testati in varie applicazioni: la rivelazione di DNA della M. tuberculosis tramite sonde a PNA, la rivelazione di DNA della fibrosi cistica tramite sonde a DNA, la rivelazione di esplosivi in traccia e la rivelazione del batterio L. pneumpohila. L’ottimizzazione delle procedure di sensing e di preparazione della superficie plasmonica, e i risultati ottenuti hanno dimostrato l’estrema versatilità dei sensori realizzati nei confronti di molteplici applicazioni sensoristiche anche molto diverse tra loro. Questo traguardo è da considerarsi un ottimo punto di partenza per studi futuri finalizzati all’ingegnerizzazione e miniaturizzazione di un sensore adattabile a diverse esigenze e applicazioni che spazino dall’area biomedica a quella della sicurezza, passando per l’analisi del cibo e dell’inquinamento.

Le zone aride sono tra le aree più sensibili al cambiamento globale e i modelli prevedono un incremento della loro superficie nei prossimi decenni. La morfologia del terreno ha un ruolo chiave nella distribuzione dell'acqua e delle sostanze nutritive nelle zone aride e nella determinazione della loro composizione. Questi ambienti sono composti da vegetazione e suolo nudo, molte volte colonizzato da biocroste, che si prevede subiranno cambiamenti nella composizione. Il telerilevamento è stato evidenziato come uno strumento importante per il monitoraggio delle zone aride. Si tratta di un approccio molto efficace in termini di costi per identificare gli hotspot di biodiversità, prevedere i cambiamenti nella loro composizione e valutare le relazioni che tali cambiamenti hanno con la morfologia del terreno. Utilizzando specifiche tecniche di analisi delle immagini a seconda del caso di studio, il telerilevamento si è dimostrato utile per il monitoraggio di zone aride ben differenziate, ma non in caso di composizione mista. Pertanto, l’obiettivo principale di questa tesi di dottorato è stato quello di studiare come la composizione eterogenea e il funzionamento delle zone aride sono influenzati dalla morfologia del terreno integrando l’utilizzo di diversi sensori di telerilevamento e piattaforme. Sono stati utilizzati dati provenienti da immagini RGB, termiche ad infrarosso (TIR), multi- e iperspettrali ad altissima risoluzione spaziale acquisite in laboratorio e in campo utilizzando piattaforme aeree, UAV e stazionarie. Sono stati definiti i seguenti obiettivi specifici: - Valutare se le tecniche Structure from Motion (SfM) possono essere utilizzate in zone aride dalla superficie complessa per ricavare la morfologia del terreno da immagini UAV; - Sviluppare una tecnica riproducibile per mettere in relazione le azioni antropiche con i cambiamenti nello stato di salute delle comunità vegetali in ecosistemi aridi utilizzando tecniche di analisi object-based; - Valutare se l'eterogeneità spettrale dei licheni può essere utilizzata per stimare la loro α-diversità utilizzando immagini iperspettrali; - Sviluppare una metodologia per valutare l’influenza della morfologia del terreno sulla distribuzione delle biocroste in zone aride utilizzando informazioni acquisite esclusivamente mediante UAV; - Valutare se le immagini TIR possono essere usate per stimare l'umidità del suolo in zone aride eterogenee. Questa tesi di dottorato comprende una valutazione delle tecniche SfM a diverse scale e della loro applicabilità a diversi livelli. Affronta lo sviluppo di una nuova metodologia per monitorare la vegetazione in un ecosistema dipendente dalle acque sotterranee, dove la loro salute è fondamentale per il funzionamento dell'ecosistema. Inoltre, l'utilizzo di immagini iperspettrali acquisite a distanza ravvicinata ha permesso di stimare la α-diversità dei licheni che formano le biocroste utilizzando la loro diversità spettrale. Questo ha portato ad una migliore comprensione del comportamento spettrale delle biocroste a seconda della loro composizione, permettendo di sviluppare una metodologia per produrre mappe accurate della copertura del suolo in un ecosistema eterogeneo e di relazionare l'effetto della morfologia del terreno sulla composizione degli ambienti aridi.
Drylands are among the most sensitive areas to actual global change and their cover will increase in the next decades. Terrain has a key role in the distribution of water and nutrients in drylands and shaping their composition. These environments are composed by vegetation and bare soil, many times colonized by biocrusts, which are expected to suffer compositional changes. Remote sensing has been highlighted as an important tool for dryland monitoring. It is a very cost-effective approach to identify biodiversity hotspots, predict changes in their composition, and to evaluate the relationships these changes have with the terrain. Using the proper image analysis according to the study case, remote sensing has proved to be useful for monitoring well differentiated drylands, but not when dryland components are mixed. Thus, the main aim of this dissertation was to study how heterogeneous dryland composition and functioning is affected by the terrain using different multiple remote sensing sensors and platforms. Data from very high spatial resolution RGB, thermal infrared, multi- and hyperspectral imagery, retrieved in the laboratory and in the field using airborne, UAV and stationary platforms were used. The next specific objectives were set: - Evaluating whether SfM techniques can be used in drylands with complex surfaces to derive their terrain from UAV imagery; - Developing a reproducible technique to relate human actions to changes in the health of dryland scarce vegetation communities by using object-based image analysis; - Testing whether the spectral heterogeneity of lichens can be used to estimate their α-diversity using hyperspectral imagery; - Developing a methodology to evaluate the control that terrain has on dryland biocrusts’ distribution using information solely retrieved from UAV; - Testing if TIR imagery can estimate soil moisture in heterogeneous drylands. This PhD thesis comprises an evaluation of SfM techniques at different scales and their applicability at different levels. It also comprises a novel methodology to monitor vegetation in a ground-water dependent ecosystem, where their health is key for the ecosystem’s functioning. Moreover, the application of close-range hyperspectral imagery allowed to estimate the α-diversity of biocrust-forming lichens using their spectral diversity. This led to a better understanding of the spectral behaviour of biocrusts depending on their composition and allowed to develop a methodology to produce accurate maps of land cover in a dryland ecosystem of heterogeneous composition and to relate the effect of terrain atrributes on dryland composition.

My PhD Thesis work, developed in Veneto Nanotech Laboratories (Nanofab in Marghera, LaNN in Padova and ECSIN in Rovigo), was aimed at the exploitation of the Surface Plasmon Resonance (SPR) phenomenon for the set-up of biosensing platforms for clinical and environmental applications. In particular, two types of SPR-based platforms were set-up and optimised: the first one was an oligonucleotide-based platform for the detection of Cystic Fibrosis (CF) causing mutations while the second one was an antibody-based platform for the detection of Legionella pneumophila whole cells. Both sensors are based on the same detection strategy, exploiting the advantages of using a highly sensitive Grating Coupled - Surface Plasmon Resonance (GC-SPR) enhanced spectroscopy method, designed using a conical illumination configuration for label-free molecular detection. Concerning DNA platform for Cystic Fibrosis, a strategy for the detection of some of the most frequent mutations responsible for CF among the Italian population is investigated. For the detection of the CF mutations, gold sinusoidal gratings are used as sensing surfaces, and the specific biodetection is achieved through the usage of allele specific oligonucleotide (ASO) DNA hairpin probes, designed for single nucleotide discrimination. Substrates were used to test unlabeled PCR amplified homozygous wild type (wt) and heterozygous samples (wt/mut) - deriving from clinical samples - for the screened mutations. Hybridisation conditions were optimised to obtain the maximum discrimination ratio (DR) between the homozygous wild type and the heterozygous samples. SPR signals obtained from hybridising wild type and heterozygous samples showed DRs able to identify univocally the correct genotypes, as confirmed by fluorescence microarray experiments run in parallel. Furthermore, SPR genotyping was not impaired in samples containing unrelated DNA, allowing the platform to be used for the parallel discrimination of several alleles also scalable for a high throughput screening setting. Concerning antibody platform for Legionella pneumophila bacteria detection, a strategy for the exploitation of the SPR phenomenon to develop a fully automated platform for fast optical detection of Legionella pneumophila pathogens was investigated. The legal limit of L. pneumophila in a high-risk hospital environment in Italy is 102 CFU/L, and the gold standard for its identification is a time consuming microbiological culture method, that requires up to 7 days. Starting from these considerations a sensitive GC-SPR system was applied to the detection of L. pneumophila to test the detection limit of the developed sensing device in term of detectable bacterium CFU. The detection was accurately set up and precisely optimised firstly through the usage of flat gold functionalised slides to be then translated to sinusoidal gold gratings for label-free GC-SPR detection using ellipsometer, in order to ensure a reproducible and precise identification of bacteria. Through azimuthally-controlled GC-SPR, 10 CFU were detected, while in the case of fluorescence analysis results, a negative readout is obtained if incubating less than 104 CFU. Successful results were obtained when incubating environmental derived samples. This detection platform could be implemented as a prototype in which water and air samples will be sequentially concentrated, injected into a microfluidic system, and delivered to the SPR sensor for analysis. The peculiar Grating Coupled - Surface Plasmon Resonance method applied for this work has therefore revealed to be an accurate and highly sensitive strategy – with multiplexing possibility - for the sensing and detecting of different kind of biomolecules, from DNA fragments to whole bacteria cell.
Il mio lavoro di Tesi di Dottorato, sviluppato presso i laboratori Veneto Nanotech (Nanofab a Marghera, LaNN a Padova ed ECSIN a Rovigo), ha avuto come obiettivo l’utilizzo della tecnologia di risonanza plasmonica di superficie (SPR – Surface Plasmon Resonance) per lo sviluppo di piattaforme biosensoristiche per applicazioni clinica ed ambientali. In particolare, durante il lavoro di Dottorato sono state messe a punto due piattaforme SPR: la prima piattaforma utilizza sonde oligonucleotidiche a DNA per l'individuazione di mutazioni causanti fibrosi cistica (CF) mentre la seconda utilizza anticorpi per il rilevamento di cellule di Legionella pneumophila. Entrambi i sensori sono basati sulla stessa strategia di rilevamento, ovvero l’utilizzo di una metodologia Grating Coupled – Surface Plasmon Resonance (GC-SPR) progettata utilizzando una configurazione conica di illuminazione ad azimut rotato per la rilevazione diretta – senza passaggi di marcatura, label-free – dell’analita in esame. Per quanto riguarda la piattaforma a DNA per la fibrosi cistica, si è sviluppata una strategia per l'individuazione di alcune delle mutazioni più frequenti responsabili CF tra la popolazione italiana. Per la rilevazione di tali mutazioni le superfici di analisi utilizzate sono grigliati sinusoidali, e la rilevazione specifica delle sequenze di interesse si ottiene attraverso l'utilizzo di oligonucleotidi allele-specifici (ASO – allele specific oligonucleotide) con struttura ad hairpin, disegnati per la discriminazione di un singolo nucleotide. I substrati plasmonici sono stati utilizzati per testare campioni wild-type ed eterozigoti (wt/mut) per le mutazioni in esame, amplificati tramite PCR a partire da campioni clinici. Le condizioni di ibridazione sono state ottimizzate per ottenere il rapporto di discriminazione (DR – discrimination ratio) massimo tra campioni wild-type ed eterozigoti. I segnali SPR ottenuti ibridando campioni wild-type e campioni eterozigoti hanno mostrato DR in grado di identificare univocamente i genotipi corretti, come confermato da esperimenti di fluorescenza in microarray eseguiti in parallelo. Inoltre la genotipizzazione ottenuta tramite SPR non è stata inficiata in campioni contenenti DNA interferente, consentendo quindi di utilizzare la piattaforma per la discriminazione in parallelo dei diversi alleli, e la possibilità futura di scalare il sistema con un approccio di high throughput screening. Per quanto riguarda la piattaforma ad anticorpi per la rilevazione di Legionella pneumophila, la medesima strategia basata su GC-SPR è stata messa a punto per ottenere una rilevazione rapida e sensibile di tale patogeno. Il limite legale di L. pneumophila in ambienti ospedalieri ad alto rischio in Italia è di 102 UFC/L (unità formanti colonia) e la metodologia di riferimento per la sua identificazione è una tecnica di coltura microbiologica che richiede tempi di attesa fino a 7 giorni. Partendo da tali considerazioni un sistema GC-SPR altamente sensibile è stato sviluppato ed applicato per la rivelazione di L. pneumophila: la rivelazione è stata accuratamente impostata ed ottimizzata con un ceppo standard del battere, prima attraverso l'utilizzo di superfici d’oro non nanostrutturate (flat) opportunamente funzionalizzate ed analizzate tramite fluorescenza, e successivamente attraverso reticoli sinusoidali (grating) d’oro analizzati tramite elissometria GC-SPR. Attraverso la metodologia GC-SPR ad azimut rotato è stato possibile rilevare fino a 10 UFC, mentre con l’analisi in fluorescenza non è stato possibile identificare quantitativi di battere inferiori a 104 UFC. Risultati positivi sono stati ottenuti anche incubando campioni di L. pneumophila isolati direttamente dall’ambiente ospedaliero. Questa piattaforma di rilevazione potrà essere implementata come prototipo in cui campioni di acqua e aria potranno venir sequenzialmente concentrati, iniettati in un sistema di microfluidica, ed incubati sulla superficie del sensore SPR per l'analisi, obiettivi questi del progetto POSEIDON (Horizon2020) attualmente in corso. La particolare metodologia GC-SPR ad azimut rotato applicata in questo lavoro di Tesi si è dimostrata essere una strategia accurata e altamente sensibile - con possibilità di multiplexing - per la rilevazione di diversi tipi di biomolecole, a partire da frammenti di DNA fino ad intere cellule batteriche.

Con l’avvento dell’Antropocene, una nuova epoca geologica caratterizzata dall’impatto delle attività umane sul clima e l’ambiente, il sistema Terra si trova a fronteggiare un netto aumento di fattori di stress di diversa origine. Tra tutti gli ecosistemi del pianeta, quelli delle regioni costiere sono sicuramente tra i più dinamici e vulnerabili. Questi sono caratterizzati da marcate dinamiche spazio-temporali e la loro posizione tra l’interfaccia terra acqua li rende soggetti ad impatti sia di origine terrestre che marina. Le minacce derivanti dai cambiamenti climatici e da un diretto impatto antropico possono danneggiare questi ambienti sia a livello locale che globale causando la perdita di habitat e un conseguente incremento della loro frammentazione. Questi disturbi possono portare a profonde trasformazioni e cambiamenti nella struttura delle comunità e, se ripetuti più volte in un breve periodo di tempo, causano una riduzione del potenziale degli ecosistemi di recuperare dopo impatti di diversa entità. In questo scenario sono essenziali valide e ripetibili tecniche di monitoraggio e mappatura per permettere di identificare e quantificare gli stress sia di natura antropica che climatica e i loro effetti sugli ecosistemi costieri. L’uso del telerilevamento per la raccolta dati rappresenta una valida soluzione per ottenere informazioni sinottiche degli ambienti impattati su diverse scale spazio temporali. Considerando queste necessità lo scopo principale del progetto di dottorato è stato quello di proporre nuovi protocolli di monitoraggio per la raccolta e l’analisi di dati da telerilevamento in regioni costiere, integrando l’uso di piattaforme e tecniche di processamento innovative. Questa ricerca descrive nuove prospettive per la raccolta di dati attraverso diverse scale temporali e spaziali usando piattaforme aeree e sottomarine. Satelliti, droni, fotogrammetria subacquea e tecniche di rilevamento acustico sono stati utilizzati per la raccolta dati in regioni costiere sia tropicali che temperate. Le informazioni raccolte sono state processate usando algoritmi sviluppati di recente come Structure from Motion (SfM), Object Base Image Analysis (OBIA) e Machine Learning. Gli articoli scientifici prodotti durante il progetto di dottorato hanno dimostrato l’elevato potenziale derivato dall’integrazione di differenti piattaforme e di metodologie di processamento dei dati. I protocolli descritti negli studi presenti in questa tesi illustrano pratiche innovative e ripetibili per la raccolta ed analisi di dati in aree costiere vulnerabili al fine di per poter valutare e quantificare gli impatti di natura antropica e climatica. I prodotti generati dalle analisi evidenziando l’occorrenza di mutamenti all’interno delle comunità e permettono di tracciare il loro declino o il potenziale recupero in un’ottica di monitoraggio e di sviluppo strategie di intervento e protezione.
The Earth system, with the entering in the new Anthropocene Epoch, is facing increasing impacts from multi-sources. Among all the environments, coastal regions are the most vulnerable, dynamic and rapidly evolving systems on the planet. Moreover, for their position at the interface between sea and emerging lands, these ecosystems are characterised by substantial spatial and temporal variability and are exposed to the impacts of both terrestrial and marine origin. Threats from climate change and direct human disturbances can affect at a regional or global scale causing habitat loss and increases of the level of fragmentation. These disturbances can lead to severe transformations, and communities shift that can be linked to the reduction of the potential of natural ecosystems to recover from multiple stressors. Under the described scenarios valid and repeatable monitoring and mapping techniques are essential to identify and quantify anthropogenic or climatic stress and their effects on coastal environments. The use of remote sensing platforms can represent a valid solution to obtain synoptic spatiotemporal data of threatened environments. According to this necessity, the primal aims of this doctoral project have been to propose monitoring protocols for collecting and analysing remote sensing data in coastal regions around the world, integrating innovative platforms and processing techniques. This research provides new insights into remote data collection and elaboration on critical coastal environments through different spatial and temporal scales. Above and underwater sensing platforms like Satellite, Unmanned Aerial Vehicles (UAVs), underwater photogrammetry and multibeam echosounder were used to collect data, and the retrieved information was processed applying recently developed algorithms such as Structure from Motion, Object Base Image Analysis and Machine Learning. The publications realised during the PhD project confirmed the high potential of the integration of different platforms and processing methodologies. The produced protocols describe innovative practices for collecting and analysing data in coastal regions in order to asses pressing anthropogenic and climatic impacts. Besides, the outputs generated from the analyses allow to highlight the occurrence of communities shift and tracking subsequent recovery or decline; they will be useful to monitor the response of the environments and address future protection strategies.