Malaria 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. / published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy
| Identifer | oai:union.ndltd.org:HKU/oai:hub.hku.hk:10722/185519 |
| Date | January 2012 |
| Creators | Cheung, Yee-wai, 張綺蕙 |
| Publisher | The University of Hong Kong (Pokfulam, Hong Kong) |
| Source Sets | Hong Kong University Theses |
| Language | English |
| Detected Language | English |
| Type | PG_Thesis |
| Source | http://hub.hku.hk/bib/B48199187 |
| Rights | The author retains all proprietary rights, (such as patent rights) and the right to use in future works., Creative Commons: Attribution 3.0 Hong Kong License |
| Relation | HKU Theses Online (HKUTO) |
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