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2012/2013 Lo sviluppo di nano-dispositivi per la misura di molecule biologiche e non biologiche è uno dei campi più importanti e in via di sviluppo della nanotecnologia. La possibilità di studiare cellule in vivo e di capirne le caratteristiche a livello molevolare ha contribuito enormemente all'avanzamento degli studi biologici negli ultimi decenni. Fra le tecniche più utilizzate, le Scanning Probe Microscopies (SPM) hanno una speciale rilevanza: sono non distruttive, con una bassa perturbazione dei sistemi studiati e permettono misure in aria e in liquido. Tali tecniche tuttavia non permettono il riconoscimento chimico, aspetto importante nello spiegare molti meccanismi cellulari. Una delle spettroscopie più utilizzate a tale scopo è il Raman, che permette il riconoscimento delle specie chimiche senza danneggiare i campioni ed è stato ampiamente utilizzato in molti studi biologici. Una combinazione di una tecnica SPM e della spettroscopia Raman è il Tip Enhanced Raman Spectroscopy (TERS): la stessa punta è usata per ricostruire le proprietà meccaniche e per illuminare localmente il campione per estrarne informazioni chimiche. Tale combinazione rende il TERS uno strumento molto potente per lo studio di strutture nanometriche. In questo lavoro di tesi abbiamo esplorato la possibilità di realizzare una punta TERS basata su un nanofilo in materiale semiconduttore cresciuto epitassialmente sulla punta di un cantilever per AFM. Il dispositivo, compatibile con le strumentazioni AFM disponibili sul mercato, ha come scopo lo sfruttamento della risonanza plasmonica creata nella particella metallica presente sulla cima del nanofilo: il segnale Raman può essere potenziato sfruttando il campo elettrico molto intenso creato in questo modo. | 910 | Abstract Nel primo capitolo si introduce le basi della tecnica AFM e vengono presentati due studi compiuti su cellule mesoteliali tramite questa microscopia. Nel secondo capitolo vengono presentati i nanofili in silicio: sono riportati i risultati ottenuti nella crescita di tali strutture, quindi vengono analizzate la citotossicità e la proliferazione di cellule su substrati di nanofili di silicio. Misure di microscpettroscopia Raman su nanofili di GaAs sono riportate nel capitolo 3: è stato studiato il cambiamento di struttura cristallina di tali nanofili dovuta a procedimenti di riscaldamenti controllati. Il capitolo 4 affronta lo studio delle proprietà SERS di nanofili di materiali e strutture diverse; sono presentati risultati sperimentali e calcoli ottenuti tramite simulazioni ad elementi finiti (FEA). Infine nel capitolo 5 presentiamo un innovativo procedimento per la fabbricazione di punte TERS a singolo nanofilo. In Appendice sono presentate brevemente le tecniche fabbricative usate durante il lavoro di tesi. The development of nano-devices for sensing molecules, both biological and non-biological is one of the most important and thriving fields of nanotechnology. The possibility to investigate living cells and their characteristics at a molecular level contributed to the great advance of biological studies in the last decades. Among the most investigated techniques, Scanning Probe Microscopies (SPM) have a prominent position: non-destructiveness, low perturbation of the sample, possibility of measurements in air and in liquid make them perfectly suited to biological studies. These techniques however lack chemical recognition of the analysed surfaces. Many cell mechanisms can be explained only understanding the chemistry involved: the integration of a spectroscopic technique is therefore essential to have insights on the phenomena under study. One of the most investigated spectroscopy for such an integration is Raman: its interaction with molecular and crystal structures allows for chemical recognition. It has been widely used in studies of organic samples an biosensing and it provides non-destructive measurements. A smart combination of a SPM technique and Raman Spectroscopy is the so-called Tip Enhanced Raman Spectroscopy (TERS) where the same probe that is used to reconstruct the sample mechanical properties with nm resolution is also used to illuminate locally the sample to extract chemical information. The high resolution spectroscopy combined with the topography of the SPM makes TERS a powerful tool for the investigation of nanometrical features. At the present, however, no commercially available TERS probe can provide reliable and reproducible results with high Raman enhancement.8 | Abstract With this thesis work we explored the realization of a TERS probe based on a semiconductor NW grown epitaxially on the apex of an AFM cantilever. This design, compatible with the commercially available equipment, aims at the use of the plasmonic resonance created in the noble metal nanoparticle present on the top of the nanowire to greatly enhance the Raman signal. The high aspect ratio of this nanostructure can lead to high-resolution topography and spectroscopy. We will first introduce the basics of SPM and give an example of a study of cells by AFM in Chapter 1: the effect of the uptake of asbestos fibers and carbon nanotubes by mesothelial cells is reported. In Chapter 2 we briefly present the growth mechanics for Si nanowires, with a summary of the results obtained for Si NWs. A study of the citotoxicity of ZnSe and Si nanowires follows: living cells were seeded on nanowires and their proliferation, behaviour and adhesion was measured as a mean to verify the compatibility of NWs with the imaging of living cells. Chapter 3 reports Raman measurements of GaAs NWs and the change in crystal structure of these upon annealing at high temperatures. Studies of the SERS effect on semiconductor nanowires are presented in Chapter 4: the plasmonic resonance on ZnSe, Si and GaAs NWs was modelled by Finite Element Method. The absorption spectra and the Raman enhancement factor of the same wires were measured and an understanding of their SERS capability is presented. Finally we propose in Chapter 5 a novel fabrication process for the localized growth of NWs on AFM cantilevers as TERS tips. Appendix A reports a brief explenation of the fabrication processes mention along the thesis presentation. XXVI Ciclo 1984

Nanotoxicology and nanomedicine investigations often require the probing of nano-objects such as fibres and particles in biological samples and cells, whilst internalization and intracellular destiny are the main issues for in vitro cellular studies. Various high resolution microscopy techniques are well suited for providing this highly sought-after information. However, sample preparation, nanomaterial composition and sectioning challenges make it often difficult to establish whether the fibres or particles have been internalized or they are simply overlaying or underlying the biological matter. In this paper we suggest a novel suitable combination of two different microscopic techniques to reveal in intact cells the uptake of asbestos fibres by mesothelial cells. After exposure to asbestos fibres and fixation, cells were first analysed under the AFM instrument and then imaged under the TwinMic soft X-ray microscope at Elettra Sincrotrone. The suggested approach combines standard soft X-ray microscopy imaging and AFM microscopy, with a common non-invasive sample preparation protocol which drastically reduces the experimental uncertainty and provides a quick and definitive answer to the nanoparticle cellular and tissue uptake.

A key challenge for the development of plasmonic nanodevices is their integration into active semiconducting structures. Gold-catalysed semiconductor nanowires are promising candidates for their bottom-up growth process that aligns a single gold nanoparticle at each nanowire apex. Unfortunately these show extremely poor plasmonic properties. In this work, we propose a way to enhance their plasmonic resonance up to those of ideal and isolated gold nanoparticles. A suitable purification protocol compatible with GaAs and ZnSe molecular beam epitaxy of nanowires is used to produce plasmonic active nanowires, which were used to enhance the Raman signal of pentacene and graphene oxide. Enhancement factors up to three orders of magnitude are demonstrated. This journal is