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|Autori: ||Cavaleri, Felicia|
|Titolo: ||Printing methodologies for functional bioarrays|
|Abstract: ||In this thesis work, solution dispensing techniques, such as inkjet printing and dip pen nanolithography, have been employed for the realization of high resolution functional bioarrays in order to study intermolecular interactions in confined environments and microarray format.
In particular, inkjet printing was employed for the generation of picoliter-scale aqueous droplets stabilized against evaporation and molecular leakage by oil-confinement with mild surfactants to artificially reproduce scalable cellular-like compartments on a chip, realizing specialized small-volume systems to study the behaviour of interacting biomolecules. In this regards we show an unprecedented solution-based protein-binding assay based on arrays of oil-confined water droplets containing protein targets and labelled ligands. Detection of few molecular binding events in these compartments is obtained by employing the advanced fluorescence fluctuation technique Raster Image Correlation Spectroscopy, here employed to probe protein-ligand interactions in artificial aqueous droplets by mapping concentration and diffusion coefficients of fluorolabeled ligands at nanomolar concentrations with a femtoliter scale resolution. RICS was used for the first time to follow molecular dynamics and binding events within confined and scalable artificial single aqueous droplets. We called this new methodology CADRICS for Confined Aqueous Droplet Raster Image Correlation Spectroscopy.
It also described a novel printing approach to produce, for the first time, stable fL-scale aqueous droplets injected inside mineral oil for studying molecular confinement and crowding effects. We printed fL aqueous droplets into oil drops on solid substrates by a field-free approach, i.e. in absence of external electric fields and electrolytes, in which we designed a novel actuating waveform by picoliter sized nozzles. Printed fL droplets form an almost-regular circular pattern at the border of mineral oil drops, given their negligible frictional force in mineral oil phase; furthermore, molecules in such fL scale compartments form ring patterns at the surfactant/oil interface due to spontaneous adsorption phenomena at the interface which, bring to molecular concentration at the drop border. At the single droplet level, we show that molecular confinement leads to modify solute-solvent and solvent driven solute-solute interactions, resulting to a decreasing of fluorescence lifetime of environment-sensitive molecular systems, such as Streptavidin-Biotin or FITC dye, but not to a significant increasing of local viscosity-sensitive molecules (CCVJ dye) or environment insensitive dyes (Alexa dyes).
Confinement at ring pattern also leads to molecular crowding, likely due to co-adsorption at the aqueous/oil interface of biomolecules and surfactants. We exploit such confinement process by a model DNA molecular machine, finding out that fluorescence signal switching-on is triggered at lower DNA target concentrations with respect to macrovolumes, thus interaction is favored in confined and crowded conditions.
The final part of the thesis is focused on a strategy for the deposition of single-stranded oligonucleotide sequences on two different solid surfaces, glass and nylon, in form of ordered arrays, through Dip Pen Nanolithography, a contact printing method to dispense drops on femtoliter scale on solid supports. The spot size on micrometer and nanometer scales strictly depends on factors such as time of contact between tip and surface, humidity, and viscosity of molecular ink. The immobilized DNA sequence is succesfully hybridized with a complementary sequence labeled with a fluorophore. The resulting double-strand DNA molecule is suitable as specific molecular recognition substrate for human Topoisomerase.|
|In||Area 02 - Scienze fisiche|
|CVLFLC88R50G263C-PhD_thesis_Cavaleri_Felicia.pdf||PhD_thesis_Felicia_Cavaleri||7,46 MB||Adobe PDF||Visualizza/apri
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