Polysaccharide coated upconversion nanoparticles for cell targeted imaging

The development of nanotechnology for biosensing and imaging has been accelerating in the past decade. Among various types of nanoprobes, lanthanide-doped upconversion nanoparticles (UCNPs) have unique optical properties unattainable by conventional fluorophores, including large anti-Stokes shifts, long luminescence lifetime, and excellent photostability. These make UCNPs a promising choice for emerging biological and biomedical applications. However, several key challenges exist and must be addressed to ensure successful implementation of UCNPs as next-generation bioprobes. In particular, excitation of UCNPs at present relies exclusively on lasers (such as 980 nm diode laser), which poses a safety concern for ordinary biomedical laboratories. The laser beam is also known to induce heating effect and phototoxicity that potentially affect cell viability and biological/physiological processes. Equally important is that despite various functionalisation methods demonstrated in literature, to date no standard protocol has been established to allow quantitative and comparative assessment of the functionalized UCNPs. Thus, my PhD thesis is centred at improving the suitability of UCNPs for biosensing and imaging applications by exploring strategies to overcome the above mentioned obstacles.

In my thesis, I first investigate the robustness and reproducibility of existing protocols on the synthesis and functionalization of UCNPs. Next, I design UCNPs that are able to be excited by light-emitting diode (LED) and compare their brightness with laser excitation. This lead to LED-excited upconversion microscopy, achieving sensitivity down to single UCNPs. I then optimised a facile ligand exchange protocol to coat UCNP with biocompatible polysaccharide, i.e. colominic acid. The followed conjugating UCNPs with antibodies allows them to be applied to the detection and imaging of specific biomolecules. An entire workflow has been set up to assess the number of antibody molecules attached to an individual nanoparticle, alongside the binding activity of the conjugated nanoparticles as compared to free antibody molecules. I have applied antibody-conjugated UCNPs to various sample types, including fixed cell lines, live cells, and tissue sections, in order to characterise the targeting ability in different biological settings. These new UCNPs is also explored in optical super-resolution microscopy and singleparticle tracking through collaborative projects. By filling the critical gaps specifically required for bioapplications, my thesis has demonstrated reliable and consistent UCNP bioprobes, which will play an important role in practical translation to advanced biosensing and imaging.