UPCONVERTING NANOPARTICLES FOR BIOSENSING AND BIOIMAGING

Upconverting nanoparticles (UCNPs) based on lanthanide-doped β-NaYF4 matrix are very promising optical materials that are getting an increasing scientific interest thanks to their unique optical properties. These properties make them particularly suitable for different applications, ranging from biosensing and medical diagnostic to photoactivated therapy. The major point of UCNPs is their anti-Stokes behaviour, thus the possibility to excite them with near infrared (NIR) light, characterized by fewer drawbacks than UV-Visible light in terms of absorption, scattering and autofluorescence of the biological matrixes, to obtain UV-Visible-NIR emissions. The thesis project is divided in three main sections: synthesis and characterization of UCNPs, their surface functionalization, and their applications in DNA-based biosensing. In details, the work is focused on the development of two different nanohybrid systems (excitable with NIR light) consisting of core-shell-shell UCNPs functionalized with an organic polymer and with biological recognition moiety (single-stranded DNA) and their use for Förster Resonance Energy Transfer (FRET) based biosensing and bioimaging in cells. Precisely, both the UCNP configurations are characterized by an inner core and two shells. The core and the first shell are doped with absorbing and emitting lanthanide ions, while the outer shell is undoped. The main difference lies in how the lanthanide ions are distributed in the different sections: one has the absorbing ions in the first shell and the emitting ones in the core, the other the opposite. Monodispersed UCNPs with both configurations are synthesized by a multi-step solution procedure at high temperature. The synthesis of UCNPs is supported by morphological (TEM), structural (XRD) and spectroscopic (steady-state and time resolved fluorescence spectroscopy) characterization. Both UCNP configurations are properly surface modified with a suitable polymer to make them dispersible in a biological medium and able to be decorated with DNA strands for FRET-based final applications. The poly(sodium 4-styrenesulfonate) polymer (PSS) is selected because among the four molecules evaluated in this thesis it is the one that better protected the nanoparticle minimizing the upconversion quenching. The last part of the work, consisting in several FRET experiments by fluorescence spectroscopy and fluorescence microscopy, has been carried out at the Université de Rouen-Normandie within the Erasmus+ traineeship programme (April-May 2023). Here, a simple proof-of-concept two-piece UCNP-based FRET biosensor is developed and evaluated also in more complex system such as human cells. The FRET-pair consists of UCNP functionalized with a single stranded DNA (working as a capture DNA, cDNA) and a specific complementary single stranded DNA decorated with a proper fluorophore (A) (referred as report DNA, rDNA). The advanced spectroscopic characterization carried out reveals that the two UCNP configurations show important differences in terms of optical properties: those with emitter ions in the inner core are generally brighter, while those with emitting ions in the shell are more sensitive for FRET-based biosensing detection of cancer biomarkers such as short oligonucleotides.