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|Arrabito, Giuseppe Domenico
|Micro and Nano patterns for Biosensing: from enzymatic assays to single cells interaction arrays
|In this thesis work, solution dispensing techniques have been employed for the realization of complex biological arrays. Inkjet printing techniques were employed for the generation of drug screening platforms. This approach was initially proved with a model enzyme system like Glucose Oxidase substrate covalently linked to a functionalized silicon oxide support. On this support an enzymatic substrate (D-glucose)/inhibitor (D-glucal) couple was accurately dispensed. A simple optical detection method was used to prove the screening capability of the microarray with the possibility to assay with high reproducibility at the single spot level. Afterwards, this methodology has been extended to CYP450 enzymes like CYP3A4, one of the main targets for the phase I drug metabolism via a droplet microreactors arrays containing CYP3A4 enzyme mixed with model inhibitors (erythromycin) and enzymatic chemiluminescent substrates (Luciferin-Isopropylacetate). The enzymatic activity was detected by using easy and low cost optical measurements of spot brightness. As a second main objective, high-throughput and multiplexed Dip Pen Nanopatterning methodologies in liquid format were combined with Proteic Ligand DNA-Directed Immobilization for the creation of complex protein biochips on modified glass surfaces displaying spots of cell-specific ligands with lateral dimensions minor than one single cell. In a first application the epidermal growth factor (EFG) protein arrays were realized to display specific single cell adhesion activity. As a second application, immobilized proteic ligands were used to recruit designed cellular receptors which presented intracellular protein domain whose interaction with a cytosolic binding partner was monitored and perturbated.
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