UCNP-based Sandwich Immunoassay for Protein Detection

Wu, Ziwei

UCNP-based Sandwich Immunoassay for Protein Detection

Wu, Ziwei

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Publication Type:: Thesis
Issue Date:: 2025

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Field	Value	Language
dc.contributor.author	Wu, Ziwei
dc.date.accessioned	2026-07-27T00:38:55Z
dc.date.available	2026-07-27T00:38:55Z
dc.date.issued	2025
dc.identifier.uri	http://hdl.handle.net/10453/195803
dc.description	University of Technology Sydney. Faculty of Science.	en_US.UTF-8
dc.description.abstract	Proteins serve as vital biomarkers across a wide range of applications, including cancer diagnostics, treatment monitoring, infectious disease surveillance, and food quality control. Sensitive and specific detection of low-abundance proteins, especially in early-stage disease or complex matrices, is critical not only for clinical decision-making and public health protection, but also for ensuring food safety and enabling large-scale biosurveillance. However, conventional detection methods such as enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) are often constrained by limited sensitivity, lengthy protocols, and reliance on bulky laboratory instrumentation, making them unsuitable for rapid and decentralized testing. To address these challenges, this thesis presents two point-of-care testing (POCT) platforms based on a sandwich immunoassay format employing upconversion nanoparticles (UCNPs) as optical reporters. UCNPs offer several advantages, including minimized autofluorescence, low background noise, and exceptional photostability, which enable high sensitivity for detecting trace levels of protein biomarkers. As a foundational step, we functionalized the UCNPs to make them water-dispersible and enable covalent antibody conjugation, ensuring their compatibility with immunoassay systems. As a foundational step, we functionalized the UCNPs with the innovated polymer to make them water-dispersible and amenable to covalent antibody conjugation, ensuring their compatibility with aqueous immunoassay systems. We first developed a lateral flow immunoassay (LFIA) employing polyclonal–polyclonal antibody pairs for the rapid and quantitative detection of A1 β-casein in milk and infant formula, using a portable reader. This platform demonstrates strong industrial potential for quality control in the dairy industry, and its sensitivity was validated against LC-MS analysis. Then, we extended the assay's applicability to a Beads-on-a-Tip platform that integrates magnetic beads (MBs)-based sandwich immunoassay with UCNPs as luminescent reporters for detecting the SARS-CoV-2 spike (S) protein. The immunocomplexes were formed in a microcentrifuge tube, enabling active washing to effectively remove unbound and non-specifically bound components, enabling active washing to effectively remove unbound and non-specifically bound components. Subsequent localized enrichment within the tip facilitates signal amplification and significantly improves detection sensitivity. Furthermore, to validate the practical applicability of the Beads-on-a-Tip platform for multiplexing detection, we demonstrated a strategy employing spectrally distinct UCNP dopants, enabling the simultaneous quantification of multiple analytes within a single assay. These capabilities significantly expand the utility of the platforms for comprehensive biomarker analysis in both clinical diagnostics and food safety applications.	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/10453/195803/1/thesis.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	© 2025 Ziwei Wu
dc.rights	au.edu.uts.lib/cph
dc.title	UCNP-based Sandwich Immunoassay for Protein Detection	en_US.UTF-8
dc.type	Thesis
utslib.copyright.status	open_access	*

Abstract:

Proteins serve as vital biomarkers across a wide range of applications, including cancer diagnostics, treatment monitoring, infectious disease surveillance, and food quality control. Sensitive and specific detection of low-abundance proteins, especially in early-stage disease or complex matrices, is critical not only for clinical decision-making and public health protection, but also for ensuring food safety and enabling large-scale biosurveillance. However, conventional detection methods such as enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) are often constrained by limited sensitivity, lengthy protocols, and reliance on bulky laboratory instrumentation, making them unsuitable for rapid and decentralized testing. To address these challenges, this thesis presents two point-of-care testing (POCT) platforms based on a sandwich immunoassay format employing upconversion nanoparticles (UCNPs) as optical reporters. UCNPs offer several advantages, including minimized autofluorescence, low background noise, and exceptional photostability, which enable high sensitivity for detecting trace levels of protein biomarkers. As a foundational step, we functionalized the UCNPs to make them water-dispersible and enable covalent antibody conjugation, ensuring their compatibility with immunoassay systems. As a foundational step, we functionalized the UCNPs with the innovated polymer to make them water-dispersible and amenable to covalent antibody conjugation, ensuring their compatibility with aqueous immunoassay systems. We first developed a lateral flow immunoassay (LFIA) employing polyclonal–polyclonal antibody pairs for the rapid and quantitative detection of A1 β-casein in milk and infant formula, using a portable reader. This platform demonstrates strong industrial potential for quality control in the dairy industry, and its sensitivity was validated against LC-MS analysis. Then, we extended the assay's applicability to a Beads-on-a-Tip platform that integrates magnetic beads (MBs)-based sandwich immunoassay with UCNPs as luminescent reporters for detecting the SARS-CoV-2 spike (S) protein. The immunocomplexes were formed in a microcentrifuge tube, enabling active washing to effectively remove unbound and non-specifically bound components, enabling active washing to effectively remove unbound and non-specifically bound components. Subsequent localized enrichment within the tip facilitates signal amplification and significantly improves detection sensitivity. Furthermore, to validate the practical applicability of the Beads-on-a-Tip platform for multiplexing detection, we demonstrated a strategy employing spectrally distinct UCNP dopants, enabling the simultaneous quantification of multiple analytes within a single assay. These capabilities significantly expand the utility of the platforms for comprehensive biomarker analysis in both clinical diagnostics and food safety applications.

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