Lanthanide Nanoparticles for Improving the Sensitivity of Mass Cytometry at the Single-Cell Level

Maddahfar, Mahnaz

Lanthanide Nanoparticles for Improving the Sensitivity of Mass Cytometry at the Single-Cell Level

Maddahfar, Mahnaz

Permalink

Publication Type:: Thesis
Issue Date:: 2022

Open Access

Copyright Clearance Process

Recently Added
In Progress
Open Access

This item is open access.

Adobe PDF

Download contents and abstractAdobe PDF (707.42 kB)

Adobe PDF

Download thesisAdobe PDF (14.3 MB)

View statistics

Full metadata record

Field	Value	Language
dc.contributor.author	Maddahfar, Mahnaz
dc.date.accessioned	2023-03-20T00:01:13Z
dc.date.available	2023-03-20T00:01:13Z
dc.date.issued	2022
dc.identifier.uri	http://hdl.handle.net/10453/167640
dc.description	University of Technology Sydney. Faculty of Science.	en_US.UTF-8
dc.description.abstract	Early detection of cancer increase the possibility of successful treatment which often requires the multiplexed detection of a panel of biomarkers of molecules and single cells. Mass cytometry (CyTOF), combining the powers of flow cytometry and mass spectrometry provides simultaneous measurement of over 40 cellular parameters at single-cell resolution, significantly augmenting the ability of cytometry to evaluate complex cellular systems and processes. This technology is based on isotopically-labelled antibodies as tags and mass spectrometry time-of-flight to distinguish the individual isotope labels on single cells. However, metal chelating polymers, currently used in CyTOF, have been found insufficient in detecting low abundance biomarkers, as number of metal atoms per tag is too low to detect biomarker expression at levels of 102 to 104 per cell. This thesis aims to address the issue of the low sensitivity of mass cytometry by developing lanthanide nanoparticles as cellular barcoding mass-tags, as individual nanoparticles can be doped with a considerable number of elemental atoms, typically in the range of 104-106 lanthanide ions per nanoparticle. As the key to producing bio-specific nanoparticles lies in the surface functionalisation of LnNPs and their subsequent conjugation to antibodies, the first focus of this thesis is on the design and synthesis of a well-defined diblock copolymer with tuneable size composed of monoacryloxyethyl phosphate block and oligo (ethylene glycol) methyl ether acrylate block through the RAFT polymerisation technique. Systematic insight into the effect of the chain length of POEGMEA on the long-term colloidal stability and antibody-conjugation efficiency of nanoparticles has been provided. Next, I explored two novel bioconjugation strategies to couple anti-B220 antibody to LnNPs: a) carbodiimide chemistry in which carboxylate groups of polymer capped LnNPs target lysine sidechains of the antibody, b) Schiff-base interaction in which hydrazide functionalised LnNPs target aldehyde groups in the Fc region of oxidised IgG antibody. Both conjugation strategies were applied to assess the sensitivity and specificity of the LnNP-coupled antibody as a ligand-specific probe for mass cytometry assays. Random orientation of antibodies on the surface of polymer-LnNP and failure to exclude free LnNPs from the coupled ones caused the Carbodimide strategy to generate significant background in CyTOF, making it difficult to distinguish signal. However, the combination of Schiff-based chemistry to orient coupling of IgG antibodies to LnNPs and the use of a blocking reagent to allow separation of free versus conjugated nanoparticles increased conjugation efficacy and significantly improved signal to noise ratio in mass cytometry assays.	en_US.UTF-8
dc.format	Thesis (PhD)
dc.language.iso	en_US	en_US.UTF-8
dc.relation	https://opus.lib.uts.edu.au/bitstream/10453/167640/2/02whole.pdf
dc.rights	info:eu-repo/semantics/openAccess
dc.rights	The author owns the copyright in this thesis including all reproduction and reuse rights for the work. The work may not be altered without the permission of the copyright owner. Attribution is essential when quoting or paraphrasing from this thesis.
dc.rights	© 2022 Mahnaz Maddahfar
dc.rights	au.edu.uts.lib/ppc
dc.title	Lanthanide Nanoparticles for Improving the Sensitivity of Mass Cytometry at the Single-Cell Level	en_US.UTF-8
dc.type	Thesis
utslib.copyright.status	open_access	*

Abstract:

Early detection of cancer increase the possibility of successful treatment which often requires the multiplexed detection of a panel of biomarkers of molecules and single cells. Mass cytometry (CyTOF), combining the powers of flow cytometry and mass spectrometry provides simultaneous measurement of over 40 cellular parameters at single-cell resolution, significantly augmenting the ability of cytometry to evaluate complex cellular systems and processes. This technology is based on isotopically-labelled antibodies as tags and mass spectrometry time-of-flight to distinguish the individual isotope labels on single cells. However, metal chelating polymers, currently used in CyTOF, have been found insufficient in detecting low abundance biomarkers, as number of metal atoms per tag is too low to detect biomarker expression at levels of 102 to 104 per cell. This thesis aims to address the issue of the low sensitivity of mass cytometry by developing lanthanide nanoparticles as cellular barcoding mass-tags, as individual nanoparticles can be doped with a considerable number of elemental atoms, typically in the range of 104-106 lanthanide ions per nanoparticle. As the key to producing bio-specific nanoparticles lies in the surface functionalisation of LnNPs and their subsequent conjugation to antibodies, the first focus of this thesis is on the design and synthesis of a well-defined diblock copolymer with tuneable size composed of monoacryloxyethyl phosphate block and oligo (ethylene glycol) methyl ether acrylate block through the RAFT polymerisation technique. Systematic insight into the effect of the chain length of POEGMEA on the long-term colloidal stability and antibody-conjugation efficiency of nanoparticles has been provided. Next, I explored two novel bioconjugation strategies to couple anti-B220 antibody to LnNPs: a) carbodiimide chemistry in which carboxylate groups of polymer capped LnNPs target lysine sidechains of the antibody, b) Schiff-base interaction in which hydrazide functionalised LnNPs target aldehyde groups in the Fc region of oxidised IgG antibody. Both conjugation strategies were applied to assess the sensitivity and specificity of the LnNP-coupled antibody as a ligand-specific probe for mass cytometry assays. Random orientation of antibodies on the surface of polymer-LnNP and failure to exclude free LnNPs from the coupled ones caused the Carbodimide strategy to generate significant background in CyTOF, making it difficult to distinguish signal. However, the combination of Schiff-based chemistry to orient coupling of IgG antibodies to LnNPs and the use of a blocking reagent to allow separation of free versus conjugated nanoparticles increased conjugation efficacy and significantly improved signal to noise ratio in mass cytometry assays.

Please use this identifier to cite or link to this item:

http://hdl.handle.net/10453/167640