During my Postdoc in US, I developed and optimized a methodology to size Extracellular Vesicles (EVs) using a home-built single molecule-sensitive flow analyser and a membrane-selective fluorescent dye (1). EVs are small lipid bilayer-enclosed structures generated and released by almost all cells in our body, and are involved in intercellular communication (exchange of proteins, lipids and genetic material). By deconvolving the intensity signal of individual vesicles from the corresponding travelling speed, I was able to improve the inter- and intra-sample accuracy and reproducibility of EVs sizing. This methodology was applied to investigate the relationship between size and proteins expression/localization on a subclass of EVs known as exosomes, which show sizes in the range of ~35-300 nm (2).
During my PhD, I focused on developing innovative fluorescent probes based on the phenomenon known as "Thermochemiluminescence" (TCL). TCL exploits a thermal triggering to generate the analytical signal, and therefore it represents an alternative to classical methodologies such as Bio- and Chemiluminescence. I developed a panel of new TCL-active substrates with tunable photophysical properties and a chemometric approach (PCA) to reveal the steric and electronic parameters that govern dioxetane formation (3). In parallel, I also developed a new nanolabel based on TCL semiconducting polymer dots (TCL-Pdots). In this label, the fluorescence emission of the Pdot is induced via a FRET mechanism by the decomposition of TCL molecules entrapped in the nanoparticle. TCL-Pdots were then properly functionalized to allow conjugation with antibodies, for immunoassay applications (4).