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postgraduate thesis: Development of aptamer-nanoparticle conjugates as a new approach to malaria diagnosis

TitleDevelopment of aptamer-nanoparticle conjugates as a new approach to malaria diagnosis
Authors
Issue Date2012
PublisherThe University of Hong Kong (Pokfulam, Hong Kong)
Citation
Cheung, Y. [張綺蕙]. (2012). Development of aptamer-nanoparticle conjugates as a new approach to malaria diagnosis. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR. Retrieved from http://dx.doi.org/10.5353/th_b4819918
AbstractMalaria is an infectious disease caused by eukaryotic protists in the genus Plasmodium. Approximately half of the world's population is at risk of malaria. The burden of Plasmodium falciparum malaria has increased in recent years due to the emergence of resistant strains, which have even been documented in regions previously reported as malaria-free. Although malaria vaccine research has been conducted and has showed recent positive results, there still remains no effective vaccine to prevent malaria in clinical practice. According to the World Health Organization, prompt confirmation of malaria infection by microscopy and/or rapid diagnostic test (RDT) is critical to control the spreading of malaria and to prevent the evolution of drug resistant Plasmodia strains. However, malaria diagnosis remains a significant challenge as many malaria endemic regions have inadequate access to microscopy, and antibody-based RDTs are restricted by their stability under tropical temperatures and by their cost. The objective of this study was to develop a new approach to malaria diagnosis using DNA aptamers to recognise proteins encoded by Plasmodium. The research is divided into two parts. Firstly, DNA aptamers against the diagnostic markers, P. falciparum histidine-rich protein 2 (HRP2) and P. falciparum lactate dehydrogenase (PfLDH), were selected by Systematic Evolution of Ligands by Exponential Enrichment (SELEX). Secondly, a selected PfLDH aptamer was incorporated into a gold nanoparticle detection system to develop an aptamer-nanoparticle conjugate as a new approach towards malaria diagnosis. The identified HRP2 and PfLDH aptamers were characterised by isothermal titration calorimetry (ITC) for their affinity to targets and were observed to bind with nanomolar affinity. As PfLDH aptamers were observed to have a higher affinity to their target, PfLDH, their specificities were further characterised by ITC using human lactate dehydrogenases, hLDHA1 and hLDHB. The PfLDH aptamers were shown to be highly specific to PfLDH with no observed affinity to human LDHs. After further characterisation, PfLDH aptamer 2008s was chosen for the next stage of the research to be combined with a nanoparticle as a route towards diagnostic application. In the second part of this study, PfLDH aptamer 2008s was conjugated to gold nanoparticles (AuNPs) to create aptamer-AuNP conjugates (2008s-AuNP). The aptamer-AuNP conjugates were characterised by their tolerance in different pH and salt concentration and in their sensitivity to PfLDH. This new approach of malaria diagnosis was further validated by incubating the aptamer-AuNP conjugates with various proteins and colour changes were observed specifically upon incubation with PfLDH but not with other proteins. Hence, a Plasmodium specific aptamer-AuNP conjugate to the malaria diagnostic marker, pLDH, has been developed in this research. This work lays the foundation for further development of novel rapid diagnostic tests based on nucleic acid aptamers and nanotechnology for robust and cost-effective malaria diagnosis with potential benefit not only for malaria but in a plethora of diagnostic applications.
DegreeDoctor of Philosophy
SubjectOligonucleotides.
Nanoparticles.
Malaria - Diagnosis.
Dept/ProgramBiochemistry
Persistent Identifierhttp://hdl.handle.net/10722/185519

 

DC FieldValueLanguage
dc.contributor.authorCheung, Yee-wai-
dc.contributor.author張綺蕙-
dc.date.accessioned2013-08-08T04:12:45Z-
dc.date.available2013-08-08T04:12:45Z-
dc.date.issued2012-
dc.identifier.citationCheung, Y. [張綺蕙]. (2012). Development of aptamer-nanoparticle conjugates as a new approach to malaria diagnosis. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR. Retrieved from http://dx.doi.org/10.5353/th_b4819918-
dc.identifier.urihttp://hdl.handle.net/10722/185519-
dc.description.abstractMalaria is an infectious disease caused by eukaryotic protists in the genus Plasmodium. Approximately half of the world's population is at risk of malaria. The burden of Plasmodium falciparum malaria has increased in recent years due to the emergence of resistant strains, which have even been documented in regions previously reported as malaria-free. Although malaria vaccine research has been conducted and has showed recent positive results, there still remains no effective vaccine to prevent malaria in clinical practice. According to the World Health Organization, prompt confirmation of malaria infection by microscopy and/or rapid diagnostic test (RDT) is critical to control the spreading of malaria and to prevent the evolution of drug resistant Plasmodia strains. However, malaria diagnosis remains a significant challenge as many malaria endemic regions have inadequate access to microscopy, and antibody-based RDTs are restricted by their stability under tropical temperatures and by their cost. The objective of this study was to develop a new approach to malaria diagnosis using DNA aptamers to recognise proteins encoded by Plasmodium. The research is divided into two parts. Firstly, DNA aptamers against the diagnostic markers, P. falciparum histidine-rich protein 2 (HRP2) and P. falciparum lactate dehydrogenase (PfLDH), were selected by Systematic Evolution of Ligands by Exponential Enrichment (SELEX). Secondly, a selected PfLDH aptamer was incorporated into a gold nanoparticle detection system to develop an aptamer-nanoparticle conjugate as a new approach towards malaria diagnosis. The identified HRP2 and PfLDH aptamers were characterised by isothermal titration calorimetry (ITC) for their affinity to targets and were observed to bind with nanomolar affinity. As PfLDH aptamers were observed to have a higher affinity to their target, PfLDH, their specificities were further characterised by ITC using human lactate dehydrogenases, hLDHA1 and hLDHB. The PfLDH aptamers were shown to be highly specific to PfLDH with no observed affinity to human LDHs. After further characterisation, PfLDH aptamer 2008s was chosen for the next stage of the research to be combined with a nanoparticle as a route towards diagnostic application. In the second part of this study, PfLDH aptamer 2008s was conjugated to gold nanoparticles (AuNPs) to create aptamer-AuNP conjugates (2008s-AuNP). The aptamer-AuNP conjugates were characterised by their tolerance in different pH and salt concentration and in their sensitivity to PfLDH. This new approach of malaria diagnosis was further validated by incubating the aptamer-AuNP conjugates with various proteins and colour changes were observed specifically upon incubation with PfLDH but not with other proteins. Hence, a Plasmodium specific aptamer-AuNP conjugate to the malaria diagnostic marker, pLDH, has been developed in this research. This work lays the foundation for further development of novel rapid diagnostic tests based on nucleic acid aptamers and nanotechnology for robust and cost-effective malaria diagnosis with potential benefit not only for malaria but in a plethora of diagnostic applications.-
dc.languageeng-
dc.publisherThe University of Hong Kong (Pokfulam, Hong Kong)-
dc.relation.ispartofHKU Theses Online (HKUTO)-
dc.rightsThe author retains all proprietary rights, (such as patent rights) and the right to use in future works.-
dc.rightsCreative Commons: Attribution 3.0 Hong Kong License-
dc.source.urihttp://hub.hku.hk/bib/B48199187-
dc.subject.lcshOligonucleotides.-
dc.subject.lcshNanoparticles.-
dc.subject.lcshMalaria - Diagnosis.-
dc.titleDevelopment of aptamer-nanoparticle conjugates as a new approach to malaria diagnosis-
dc.typePG_Thesis-
dc.identifier.hkulb4819918-
dc.description.thesisnameDoctor of Philosophy-
dc.description.thesislevelDoctoral-
dc.description.thesisdisciplineBiochemistry-
dc.description.naturepublished_or_final_version-
dc.identifier.doi10.5353/th_b4819918-
dc.date.hkucongregation2012-

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