Preparation, Surface Modification and Application of Lanthanide Nanoparticles as Elemental Tags in Mass Cytometric Bioassays

This thesis describes the preparation, surface modification and application of lanthanide nanoparticles (Ln NPs) as elemental tags in bioassays by mass cytometry. The most common elemental tags in mass cytometry are lanthanide metals. Previous studies established polymer-based lanthanide tags for mass cytometric bioassays. My studies focused on developing a new generation of elemental tags based on Ln NPs for bioassays by mass cytometry. The first part of my thesis describes a ligand exchange strategy for making Ln NPs that are colloidally stable in aqueous buffer media. This strategy yielded NaLnF4 NPs capped with PEG-PAMAM surface ligands. These PEG-PAMAM-capped NPs were colloidally stable in water but lacked long-term colloidal stability in PBS. This made the PEG-PAMAM-capped NPs incompatible with cell assays by mass cytometry. The second part describes the preparation of small LnF3 NPs as internal labels for Ln-containing polymer microspheres. This work was done by collaboration with Dr. Jianbo Tan. He prepared PMMA microspheres labeled with those small LnF3 NPs. We then developed a streptavidin-biotin binding assay using those LnF3-labeled microspheres as a proof-of-concept bead-based assay by mass cytometry. The third part describes a sandwich-type bead-based immunoassay by mass cytometry. I prepared NaYF4@SiO2 NPs as reporter tags for this bead-based immunoassay. By using the LnF3-labeled PMMA microspheres as bead supports, I established a bead-based immunoassay that specifically targets human IgG and tested the specificity of this assay by TEM. The fourth part discusses making a NP-based elemental tag that can specifically target cell surface markers with minimal non-specific cell binding. I synthesized PEGylated NaHoF4@SiO2 NPs and attached antibodies onto the PEG corona of the NPs. These NP-antibody conjugates showed excellent targeting ability for CD20 antigens on Ramos cells along with a low background level of non-specific binding. Future experiments are needed to demonstrate their usage in detecting low-abundance cellular biomarkers by mass cytometry.Ph.D