Nardon, Chiara (2010) Design and development of new metal-based biosensors for the early detection of cancer antigens in oncology. [Laurea specialistica biennale]
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Nowadays, the goal in oncological diagnostics is to tackle the growth and the spread of cancer by developing new cost-effective screening techniques for early diagnosis, in order to both improve the survival rate of patients and reduce costs related to the various prevention programmes. In an interdisciplinary context it has been designed a new method to early detect tumor biomarkers present either in blood or in urine based on the use of lanthanide (Ln) complexes. By exploiting a time-resolved technique we aim at achieving a detection limit lower than 1 U/mL and/or obtaining a faster, easier and less expensive sensing method alternative to the widely employed ELISA (enzyme linked immunosorbent assay). In fact, lanthanides offer several advantages compared to the common organic labels. The luminescence lifetime of the specific signal is several orders of magnitude longer than the non-specific background, thus enabling the label to be measured without background interference (time resolution). The large Stokes’ shift and the narrow emission peak contribute toward increasing the signal-to-noise ratio. We worked following two routes. In the first one, called CHIA (Chemiluminescence ImmunoAssay), we designed to develop a novel type of biosensors based on changes of luminescence intensity or wavelength of lanthanide complexes. We decided to investigate tryptophan (Trp) residues as possible donors in energy transfers and serine residues as possible bioconjugation sites of lanthanide complexes to the monoclonal antibodies. According to the Dexter’s mechanism, a LRET from Trp to the metal ion could occur. Since the Trp-Ln distance is a relevant factor in determining the RET effectiveness, we pointed out our attention to a specific type of antibody, as an example. We chose colo-rectal cancer as it is a quite common tumor. After having looked for the exact sequence of CA19-9 mAb and the structure of similar Fab with an high sequence homology, a study at a biochemical level of the antibody structure and the involved distances revealed the presence of many couples Trp-Ser spaced less than 10 Å. This encouraged the subsequent digestion trials of a cheap test antibody by using ficin protease yielding F(ab’)2 fragments, characterized and purified by means of SDS-PAGE and SEC. In the second approach, called LIA (Luminescence ImmunoAssay), six lanthanide complexes have been synthesised and widely characterized by using two different type of ligands: two dithiocarbamato and four coumarin-3-carboxylic acid (HCCA) derivatives. We studied luminescence emission for all complexes in acetonitrile, water and dimethyl sulfoxide.
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