Nuove strategie per la rilevazione ad alta sensibilità di analiti biologici

The activities of this PhD, done in the context of the PLATFORM strategic project of the University of Padua, dealt with the study and optimization of analytical techniques for the detection of biologically relevant analytes of different kinds, as proteins, nucleic acids, small molecules. The research relied essentially on two technological platforms currently under development in the laboratories of reference, and was carried in tight collaboration with other research groups of the University of Padua. The first platform exploited the surface plasmon resonance (SPR) for the detection of the interaction of an analyte in solution with a receptor grafted on nanostructured surfaces made of noble metals. Objective of the research was the synthesis optimization and the chemical and functional characterization of various molecules suitable for the grafting of biological receptors (as DNA or its PNA analogue) on nanostructured surfaces (sinusoidal gratings) for SPR analysis; to this purpose protocols were developed and optimized for the synthesis of different Poly(Ethylene Glycol) (PEG) derivatives dedicated to this; also, functionalization strategies were developed, for an easy and economic processing of the surfaces. Several superficial functionalization conditions were explored, finding the most performing for the analytical set-up in terms of detection capability and resistance to nonspecific interactions; finally, detection tests of biological analytes were performed employing the optimized set-up, and the results were compared with a technique currently in use, highlighting benefits and disadvantages of the studied analytical system. The second platform saw the use of the poly-avidin ANANAS® nanoparticles of recent introduction as a sensitivity amplification element of common immunoenzymatic assays, based on the coupling between the analyte-receptor interaction with the activity of enzymes capable of generating color or light quantums. Objective of the research was the exploration of several analytical configurations exploiting the nanoparticles for the detection of micro- or macro-molecular analytes in direct or indirect assays; to this purpose the synthesis and the chemical and functional characterization was pursued of all the biotinylated elements necessary to the development of analytical protocols involving the ANANAS nanoparticles, with particular regard to the realization and setting of an homogenous assay for the detection of a model analyte in solution.