Investigation into aptamer-mediated diagnosis of malaria

Abstract

Malaria is an infectious disease caused by parasitic protozoans from the genus Plasmodium, with the species P. falciparum causing the highest number of deaths worldwide and P. vivax being the most widespread. Malaria also has significant socio-economic growth penalties on endemic countries in poverty alleviation, economic growth and education. Therefore, massive resources have been poured into the management, prevention and eradication of the disease. Rapid diagnostic tests (RDTs) have become essential in this management effort, however, current RDTs that detect P. falciparum and P. vivax are primarily antibody-based, which can have significant drawbacks in cost, temperature stability and robustness.

In this thesis, I report the development of several DNA aptamer-based biosensing alternatives for malaria detection. As aptamers have been shown to be cheaper to produce, easier to modify and temperature stable. I characterized canonically selected DNA aptamers and non-natural chemical evolutionary selected benzene isostere cubane-modified aptamers for their ability to bind to P. falciparum histidine-rich protein II (PfHRP2), P.falciparum lactate dehydrogenase (PfLDH) and P. vivax lactate dehydrogenase (PvLDH).

I introduced the PvLDH targeting cubane-modified aptamer “cubamer” into enzyme-linked colourimetric assays through additional biotinylation modifications. The assays were able to successfully discriminate between the P. falciparum and P. vivax infections which was not previously possible through canonically selected aptamers. Furthermore, a visible detection limit of 123 pM was achieved, which reaches much lower than the reported clinical range for PvLDH.

The PfHRP2 targeting aptamer was integrated into an electrochemical-based biosensing platform. By modifying the aptamer with a redox reporter and attachment to gold surfaced electrodes, the developed biosensor exhibited high sensitivity, specificity and stability in target recognition when challenged against complex serum buffers. In particular, in human serum buffer a detection limit of 3.73 nM was achieved against PfHRP2. Furthermore, the biosensor was re-useable, and maintained stability when stored up to a week at room temperature.

To achieve higher sensitivity, the PfHRP2 detecting electrochemical biosensor was further integrated with Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated enzyme signal amplification. A novel “lock and key” mechanism was designed that allowed for the detection of target malaria protein through CRISPR’s ability to cleave DNA. I show that this use of CRISPR allowed for new ways to significantly reduce off-target detection signals, as well as build on the field of protein detection by CRISPR.

The findings of my thesis provide strong evidence for aptamers as promising alternatives to conventional antibody-based P. falciparum and P. vivax detection for malaria diagnosis. Future developments will focus on their implementation in the field, and bringing them closer to benefiting the global efforts in malaria management.