Development of Lanthanide Nanoparticles for High Sensitivity Mass Cytometry Immunoassay

This thesis describes the synthesis and characterization of lanthanide nanoparticle (NP) based reagents for mass cytometry (MC) immunoassays. MC is a single cell analysis technique that uses metal-based tags and inductively coupled plasma-mass spectrometry (ICP-MS) time-of-flight (TOF) detection. Currently, mass tags contain ~ 200 lanthanide (Ln) ions per antibody (Ab) conjugate. These conjugates are effective at detecting abundant surface markers (> 104 per cell). As an approach to increase the sensitivity of MC, I designed NP reagents which contain > 104 Ln metal ions per NP. In my research, I developed a series of uniform lanthanide NPs such as LaF3 and NaLnF4 (Ln: Y, Sm to Ho) with sizes between 5 to 30 nm. I optimized the reaction parameters such as the heating rate, temperature, heating time and solvent ratio for each lanthanide in order to obtain uniform NPs. In the second step, I developed two methods to render the NPs colloidally stable in water and in buffers: ligand exchange and lipid encapsulation. For ligand exchange, I used PEGylated phosphate/phosphonate ligands to transfer the NPs into water. However, these NPs showed poor colloidal stability in phosphate buffered saline (PBS). To understand this process, I quantified the ligand footprint of mono- and multidentate polyethylene glycol (PEG) phosphate ligands using three independent analytical techniques. The ligand density measurements obtained from each technique were in good agreement. For lipid encapsulation, I coated the NPs either with a lipid monolayer or a lipid bilayer. For the lipid monolayer coating, I encapsulated oleate-capped NPs with PEG lipids, but these NPs were stable in water but not in PBS. To improve the colloidal stability, I coated the NPs with a lipid bilayer. The hydrophobic NPs were modified with citrate and then treated with a ternary lipid mixture. The resulting NPs exhibited exceptional colloidal stability in both water and PBS for up to 1 month. Finally, secondary antibodies were conjugated to the NPs using the post-insertion method for specific binding to cells. The specific-to-nonspecific binding ratio of the Ab-NP conjugates evaluated with Ramos (CD20+) and HL60 (CD20-) cells was 10 to 20.