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postgraduate thesis: Gold nanoparticles as a mass tag signal amplification strategy for the sensitive detection of cervical cancer marker protein via immuno-capture laser ionization mass spectrometry
Title | Gold nanoparticles as a mass tag signal amplification strategy for the sensitive detection of cervical cancer marker protein via immuno-capture laser ionization mass spectrometry |
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Authors | |
Advisors | Advisor(s):Che, CM |
Issue Date | 2020 |
Publisher | The University of Hong Kong (Pokfulam, Hong Kong) |
Citation | Tam, T. S. [譚兆聰]. (2020). Gold nanoparticles as a mass tag signal amplification strategy for the sensitive detection of cervical cancer marker protein via immuno-capture laser ionization mass spectrometry. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR. |
Abstract | Sexually transmitted infections by high-risk types of human papillomaviruses (HPV) are responsible for most of the cervical cancer incidents in women worldwide. A current challenge is the development of assays that reliably diagnose high risk HPV infections during the early stages of infection. HPV oncoproteins E6 and E7 have been suggested as suitable biomarkers for the early diagnosis of cervical cancer given the critical role they play in carcinogenesis. Development of increasingly sensitive techniques capable of detecting the oncoprotein at trace levels would be beneficial to patient therapy and prognosis.
Here, an immuno-capture laser ionization mass spectrometry (LI-MS) assay was developed for the detection of HPV18E7 oncoprotein. Integrating solid-phase immunoassays with mass spectrometry, the immuno-capture LI-MS assay features gold nanoparticles (AuNPs) as mass tags. When subject to laser irradiation, the plasmonic property of AuNPs facilitate the generation of gold cluster ions from the surface of the nanoparticle, which are then detected via MS. Coupled with their ease of biofunctionalization, AuNPs can be modified with antibodies for the specific and sensitive analysis of target biomolecules.
In this study, an immuno-capture substrate was fabricated as part of a solid-phase sandwich immunoassay to detect HPV18E7 oncoprotein. It was found that optimizing the silanization solvent, time, and printing conditions critically influences the density of capture antibodies on substrate surface. AuNP mass tags were developed via the synthesis of 20nm AuNPs, followed by surface modification using polyethylene glycol (PEG) to attach antibodies to the AuNPs. Both nanoparticle shape (spherical vs. star-shaped), and positive/negative ionization mode were studied to determine their effects on gold cluster ion generation by AuNPs. Surface modification of AuNPs was optimized by varying ratios of antibody-capped PEG versus carboxylate-capped PEG to control the degree of specific versus non-specific binding. By modifying this ratio, an 8-fold improvement was observed for the signal-to-noise in detecting the HPV18E7 at low femtomole level.
The developed method utilizing AuNP mass tags was applied to detect native HPV18E7 in HeLa cell lysate, where the oncoprotein content was found to be 127 ± 30 attommole per 12,500 cells (10 ± 2 zeptomole per cell) via method of standard addition. These results indicate that the immuno-capture LI-MS is a sensitive technique for the detection of antigens at trace levels, and the development of nanoparticle-based mass tags may show promise for biomarker detection in solid-phase immunoassays for diagnostics and potentially other applications.
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Degree | Master of Philosophy |
Subject | Cervix uteri - Cancer - Diagnosis Gold Nanoparticles |
Dept/Program | Chemistry |
Persistent Identifier | http://hdl.handle.net/10722/282132 |
DC Field | Value | Language |
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dc.contributor.advisor | Che, CM | - |
dc.contributor.author | Tam, Toby Siu-chung | - |
dc.contributor.author | 譚兆聰 | - |
dc.date.accessioned | 2020-05-02T03:09:11Z | - |
dc.date.available | 2020-05-02T03:09:11Z | - |
dc.date.issued | 2020 | - |
dc.identifier.citation | Tam, T. S. [譚兆聰]. (2020). Gold nanoparticles as a mass tag signal amplification strategy for the sensitive detection of cervical cancer marker protein via immuno-capture laser ionization mass spectrometry. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR. | - |
dc.identifier.uri | http://hdl.handle.net/10722/282132 | - |
dc.description.abstract | Sexually transmitted infections by high-risk types of human papillomaviruses (HPV) are responsible for most of the cervical cancer incidents in women worldwide. A current challenge is the development of assays that reliably diagnose high risk HPV infections during the early stages of infection. HPV oncoproteins E6 and E7 have been suggested as suitable biomarkers for the early diagnosis of cervical cancer given the critical role they play in carcinogenesis. Development of increasingly sensitive techniques capable of detecting the oncoprotein at trace levels would be beneficial to patient therapy and prognosis. Here, an immuno-capture laser ionization mass spectrometry (LI-MS) assay was developed for the detection of HPV18E7 oncoprotein. Integrating solid-phase immunoassays with mass spectrometry, the immuno-capture LI-MS assay features gold nanoparticles (AuNPs) as mass tags. When subject to laser irradiation, the plasmonic property of AuNPs facilitate the generation of gold cluster ions from the surface of the nanoparticle, which are then detected via MS. Coupled with their ease of biofunctionalization, AuNPs can be modified with antibodies for the specific and sensitive analysis of target biomolecules. In this study, an immuno-capture substrate was fabricated as part of a solid-phase sandwich immunoassay to detect HPV18E7 oncoprotein. It was found that optimizing the silanization solvent, time, and printing conditions critically influences the density of capture antibodies on substrate surface. AuNP mass tags were developed via the synthesis of 20nm AuNPs, followed by surface modification using polyethylene glycol (PEG) to attach antibodies to the AuNPs. Both nanoparticle shape (spherical vs. star-shaped), and positive/negative ionization mode were studied to determine their effects on gold cluster ion generation by AuNPs. Surface modification of AuNPs was optimized by varying ratios of antibody-capped PEG versus carboxylate-capped PEG to control the degree of specific versus non-specific binding. By modifying this ratio, an 8-fold improvement was observed for the signal-to-noise in detecting the HPV18E7 at low femtomole level. The developed method utilizing AuNP mass tags was applied to detect native HPV18E7 in HeLa cell lysate, where the oncoprotein content was found to be 127 ± 30 attommole per 12,500 cells (10 ± 2 zeptomole per cell) via method of standard addition. These results indicate that the immuno-capture LI-MS is a sensitive technique for the detection of antigens at trace levels, and the development of nanoparticle-based mass tags may show promise for biomarker detection in solid-phase immunoassays for diagnostics and potentially other applications. | - |
dc.language | eng | - |
dc.publisher | The University of Hong Kong (Pokfulam, Hong Kong) | - |
dc.relation.ispartof | HKU Theses Online (HKUTO) | - |
dc.rights | The author retains all proprietary rights, (such as patent rights) and the right to use in future works. | - |
dc.rights | This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License. | - |
dc.subject.lcsh | Cervix uteri - Cancer - Diagnosis | - |
dc.subject.lcsh | Gold | - |
dc.subject.lcsh | Nanoparticles | - |
dc.title | Gold nanoparticles as a mass tag signal amplification strategy for the sensitive detection of cervical cancer marker protein via immuno-capture laser ionization mass spectrometry | - |
dc.type | PG_Thesis | - |
dc.description.thesisname | Master of Philosophy | - |
dc.description.thesislevel | Master | - |
dc.description.thesisdiscipline | Chemistry | - |
dc.description.nature | published_or_final_version | - |
dc.date.hkucongregation | 2020 | - |
dc.identifier.mmsid | 991044227171703414 | - |