Digital Profiling of Circulating Extracellular Vesicles at Single-Upconversion Nanoparticle Sensitivity and Resolution

Huang, Guan

Digital Profiling of Circulating Extracellular Vesicles at Single-Upconversion Nanoparticle Sensitivity and Resolution

Huang, Guan

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Publication Type:: Thesis
Issue Date:: 2022

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Field	Value	Language
dc.contributor.author	Huang, Guan
dc.date.accessioned	2022-11-28T01:42:48Z
dc.date.available	2022-11-28T01:42:48Z
dc.date.issued	2022
dc.identifier.uri	http://hdl.handle.net/10453/163768
dc.description	University of Technology Sydney. Faculty of Science.	en_US.UTF-8
dc.description.abstract	Circulating extracellular vesicles (EVs) carry significant information about the progression stages of tumour sites. Quantification of low-abundant EVs and statistical profiling of the heterogeneity of single EVs, particularly from liquid biopsy sampling, will guide clinical decisions on the stages of tumour progression. However, the nanoscopic sizes (typically 40-200 nm) and the extremely small quantity of cargo materials demand the high detection sensitivity, stability, resolution and throughput to be simultaneously achieved. Nanotechnology has been broadly used in the field of liquid biopsy. This thesis explores a new strategy for ultra-sensitive, photo-stable, and super-resolution immunoassay of single EVs, which is based on the development, bio-conjugation and application of upconversion nanoparticles (UCNPs). In chapter 2, we apply UCNPs for direct enumeration of single CD9 and EpCAM positive EVs (CD9+EpCAM+EVs). The achieved single-molecule sensitivity results in a femtomolar detection limit (1.8 × 106 EVs mL-1), which was nearly 3 orders of magnitude lower than the standard enzyme-linked immunosorbent assay (ELISA). Compared with previous luminescence resonance energy transfer (LRET) method using UCNPs for detection of EVs, our technique achieves single tumour-derived sEV quantification. In chapter 3, we report super-resolution imaging technique for single CD9+EpCAM+EV analysis. The upconversion luminescence of single UCNPs can nonlinearly response to a donut-shaped scanning beam, so that a resolution better than 40 nm can be achieved beyond the diffraction limit. In chapter 4, with the ultra- sensitivity and photo stability achieved by UCNPs as well as super resolution offered by a donut-shaped scanning, the preclinical translation capability of the integrated technology platform has been examined by two types of breast cancer mouse models. Our results suggest that the population of cancer-derived circulating EVs, detected and classified by the number of UCNPs, can be used to monitor the metastatic tumour progression, including non-metastasis/high-metastasis and low-metastasis/high-metastasis mouse models. Furthermore, we find that the number of UCNPs on single EVs can be used to index the stage of metastatic tumour progression. In chapter 5, we discuss the challenges and opportunities of this thesis towards clinical translation, which suggests a new scope of research by integrating nanotechnology, microscopy imaging and lab-on-a-chip devices for EV research and applications. This thesis presents a viable approach of using the EVs-based liquid biopsy for tumour diagnosis and prognosis.	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/10453/163768/2/02whole.pdf
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	au.edu.uts.lib/ppc
dc.rights	info:eu-repo/semantics/openAccess
dc.title	Digital Profiling of Circulating Extracellular Vesicles at Single-Upconversion Nanoparticle Sensitivity and Resolution	en_US.UTF-8
dc.type	Thesis
utslib.copyright.status	open_access	*

Abstract:

Circulating extracellular vesicles (EVs) carry significant information about the progression stages of tumour sites. Quantification of low-abundant EVs and statistical profiling of the heterogeneity of single EVs, particularly from liquid biopsy sampling, will guide clinical decisions on the stages of tumour progression. However, the nanoscopic sizes (typically 40-200 nm) and the extremely small quantity of cargo materials demand the high detection sensitivity, stability, resolution and throughput to be simultaneously achieved. Nanotechnology has been broadly used in the field of liquid biopsy. This thesis explores a new strategy for ultra-sensitive, photo-stable, and super-resolution immunoassay of single EVs, which is based on the development, bio-conjugation and application of upconversion nanoparticles (UCNPs). In chapter 2, we apply UCNPs for direct enumeration of single CD9 and EpCAM positive EVs (CD9+EpCAM+EVs). The achieved single-molecule sensitivity results in a femtomolar detection limit (1.8 × 106 EVs mL-1), which was nearly 3 orders of magnitude lower than the standard enzyme-linked immunosorbent assay (ELISA). Compared with previous luminescence resonance energy transfer (LRET) method using UCNPs for detection of EVs, our technique achieves single tumour-derived sEV quantification. In chapter 3, we report super-resolution imaging technique for single CD9+EpCAM+EV analysis. The upconversion luminescence of single UCNPs can nonlinearly response to a donut-shaped scanning beam, so that a resolution better than 40 nm can be achieved beyond the diffraction limit. In chapter 4, with the ultra- sensitivity and photo stability achieved by UCNPs as well as super resolution offered by a donut-shaped scanning, the preclinical translation capability of the integrated technology platform has been examined by two types of breast cancer mouse models. Our results suggest that the population of cancer-derived circulating EVs, detected and classified by the number of UCNPs, can be used to monitor the metastatic tumour progression, including non-metastasis/high-metastasis and low-metastasis/high-metastasis mouse models. Furthermore, we find that the number of UCNPs on single EVs can be used to index the stage of metastatic tumour progression. In chapter 5, we discuss the challenges and opportunities of this thesis towards clinical translation, which suggests a new scope of research by integrating nanotechnology, microscopy imaging and lab-on-a-chip devices for EV research and applications. This thesis presents a viable approach of using the EVs-based liquid biopsy for tumour diagnosis and prognosis.

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