Dottorato in SCIENZE FARMACEUTICHE

Titolo della tesi: Design of Surface Plasmon Resonance (SPR)-based Biosensors for antibodies and nucleic acids’ analytical detection

This PhD thesis is focused on Surface Plasmon Resonance (SPR) based biosensors used for the analytical detection of antibodies and nucleic acids and it has been divided in two main parts. The part A regards the design of new immobilization method to control the antibodies’ orientation in the development of SPR-immunosensors. Most of immobilizing methods lead to a random orientation with a lower binding site density and immunoaffinity. Resorc[4]arene derivatives have been proposed as an alternative tool for the orientation of antibodies on the sensor surface, thanks to their particular structure of lower and upper-rims which can be modified to bind specific guest molecules. SPR-technique has been used to analyze the surface coverage and the orientation of insulin antibody (Ab-Ins) showing the resorcarene-based immunosensor as a powerful system with improved sensitivity as well as new insight into sensors development. The Part B is based on the development of SPR sensor for detection at low levels in plasma samples of miRNA-16 and miRNA-125 emerged as promising diagnostic and prognostic biomarkers of myelodysplastic syndrome. Since the conventional methods for the quantification of miRNAs (RT-qPCR, microarrays, and northern blotting) require expensive and complex equipment, the SPR technique has been proposed as alternative methods emerging as the most promising for rapid, sensitive, and on-site analysis. An optical SPR biosensor based on new sandwich assay format has been developed in which the binding of functionalized gold nanoparticles on surface was used to enhance the sensor response. Moreover, in order to reduce the effect of the protein aggregated or RNAase occurred in the matrix, a new preparation method of plasma samples has been proposed and optimized.