Ottimizzazione del protocollo di detection della proteina E del Virus Dengue, tramite nanobiosensori meccanici

Abstract

For biomolecular diagnostic is essential to use devices capable of label-free detection, quantitative measurement and miniaturization technologies. Microcantilever represents one of the most simplified MEMs based devices; an extremely significant advantage in using it is the high mass resolution: zeptograms (10-18g) for operating procedure in vacuum and nanograms (10-9g) for operating procedure in liquid. The thesis work was structured in two lines of study: the first oriented to verify the entity of chemical silicon-surface functionalization; the second finalized to the development of the bioassay procedure for Dengue virus detection. Functionalization can be viewed as a three step process: first, self-assembly of an amino-terminated organic layer using 3-aminopropyltriethoxysilane (APTES). Second, conversion of APTES in APTES/SA layer. Third, Carboxyl groups on succinylated APTES are turned into amino-linker by reaction with EDC-sNHS. The development of the bioassay procedure was supported by comparison with ELISA assay, and was focused on: verifying the serotype-specific interaction among anti-DENV1 mAbs and domain III of E protein, comparison between anti-DENV1 scAb and FullAb interaction with DIII-DV1, optimization of oriented deposition of FullAb through Protein G, optimization of passivation procedure. Nanomechanical characterization of antigen-antibody interaction has permitted to quantify the DV1-DIII deposition layer, evaluate of about 1013 moleucles/cm2.