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

Abstract

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.