BIOMOLECULAR PRINTING FOR ARTIFICIAL LIFE-INSPIRED COMPARTMENTS AND SINGLE-CELL ARRAYS

In this thesis work, solution dispensing techniques at micro- scale and nano- scale, such as inkjet printing and dip-pen nanolithography (DPN), have been employed in order to develop a new class of platforms for artificial compartments fabrication and adherent cells manipulation. In particular, the first part of the work has been focused on the fabrication of artificial compartments by new approaches combining tools derived from printing techniques and solution approaches. Firstly, a new strategy to prepare suspension of femtoliter oil droplets in water by a novel inkjet printing-based method has been designed. The oil-in-water (o/w) emulsions have been obtained by printing pL-scale fluorinated oil (FC40) droplets directly into a solution of perfluoro-1-octanol as surfactant. Then, the fragmentation of the pL drops in fL droplets occurred by a Plateau−Rayleigh mechanism at the water/air interface in presence of the non-ionic surfactant Tween 80 at low concentrations (0.003% v/v). The emerging fragmented droplets were investigated in terms of size and number by an impedance-based electrical characterization chip. Secondly, a different wet chemistry approach has been defined in order to prepare subcellular artificial compartments at the nanoscale. Specifically, a new method for the preparation of pH-responsive polymersomes, artificial polymeric vesicles usable as stimuli-responsive nanocarriers for fluorescent anticancer drugs, such as doxorubicin. The polymersomes dispersions were obtained from the synthesis of the polymer components. Initially, a new class of pH-sensitive di-block copolymers of general structure mPEGxPDEAMAy-Br, were synthesized by the atom transfer radical polymerization (ATRP), a quasi-living radical polymerization reaction. The synthesized copolymers are water soluble at acidic pH ( ~ 2 ) and can self-assemble into polymeric vesicles as pH is increased, through a deprotonation mechanism at the PDEAMA block. The diffusion behaviour of the resulting vesicles (90 nm in diameter) has been investigated in solution and inside cells by means of raster image correlation spectroscopy (RICS), an advanced fluorescence-based fluctuation technique, which allows to study the dynamic behaviour of fluoro-labelled molecules and colloids. The second part of the thesis work has been focused on the fabrication of biomaterials micropatterns for applications as biosensors and biochips. A novel approach based on inkjet printing has been exploited to pattern cell-adhesive biomaterials for single-cell harvesting and manipulation. The chip was realized by a novel green chemistry approach that combines polymeric layer deposition and functionalization approach based on inkjet printing to tailor a biointerface suitable for single-cell attachment. The resulting single-cell biochip developed in this thesis consists of an array of isolated human lung cancer cells (H1975) captured on inkjet-printed collagen spots, whose lateral size (32 µm in diameter) has been specifically tailored in order to fit the dimensions of a single cell. The platform is suitable for cell growth and proliferation, and it allows cells attaching and remaining vital up to one week at least in the array format. The polymeric layers and patterns have been characterized at each step of fabrication by surface analysis techniques, including atomic force microscopy (AFM) and X-ray photoelectron spectroscopy (XPS), while the cells analysis and the materials staining protocols were investigated by means of laser scanning confocal microscopy (CLSM). Finally, a DPL-based mechanism for the imbibition of aqueous inks containing single-strand oligonucleotide sequences on nylon, a technologically relevant flexible substrate, has been investigated. The mechanism of droplets deposition on such a porous substrate has been studied on both theoretical and experimental point of view. In addition, the oligonucleotide sequences, after covalent immobilization on nylon by UV irradiation, have been hybridized with two fluoro-labelled partial complementary sequences, in order to assemble a DNA-based supramolecular structure, which can be employed for specific molecular recognition by human Topoisomerase IB, a model protein for the Plasmodium topoisomerases, which are potential biomarkers for Malaria diagnosis. This new class of DNA-based functional substrates can be considered as a first step for the point-of-care diagnostic on a biochip platform for identification of molecular disease-related protein as biomarkers.