DNA aptamers have potential for disease diagnosis and as therapeutics, particularly when interfaced with programmable molecular technology. Here we have combined DNA aptamers specific for the malaria biomarker Plasmodium falciparum lactate dehydrogenase (PfLDH) with a DNA origami scaffold. Twelve aptamers that recognise PfLDH were integrated into a rectangular DNA origami and atomic force microscopy demonstrated that the incorporated aptamers preserve their ability to specifically bind target protein. Captured PfLDH retained enzymatic activity and protein-aptamer binding was observed dynamically using high-speed AFM. This work demonstrates the ability of DNA aptamers to recognise a malaria biomarker whilst being integrated within a supramolecular DNA scaffold, opening new possibilities for malaria diagnostic approaches based on DNA nanotechnology.
We present the adaption
of an aptamer-tethered enzyme capture (APTEC)
assay into point-of-care device prototypes with potential for malaria
diagnosis. The assay functions by capturing the malaria biomarker Plasmodium falciparum lactate dehydrogenase (PfLDH) from samples and using its intrinsic enzymatic activity to generate
a visualizable blue color in response to Plasmodium positive samples. Using three-dimensional (3D) printing rapid prototyping,
a paper-based syringe test and magnetic bead-based well test were
developed. Both were found to successfully detect recombinant PfLDH at ng mL–1 concentrations using
low sample volumes (20 μL) and could function using purified
or spiked whole blood samples with facile sample preparation. The
syringe test was found to be more analytically sensitive but required
more additional preparation steps, while the well test required fewer
steps and hence may be better suited for future clinical testing.
Additionally, the development reagents required for the color response
were fully stabilized through desiccation with sugar stabilization
agents and could withstand temperatures as high as 90 °C. This
study demonstrates how a previously reported biochemical assay can
be adapted into workable point-of-care device prototypes by using
3D printing rapid prototyping. This novel technology, intended for
rapid diagnostic tests (RDTs) for malaria, is distinct and carries
many potential advantages relative to established lateral flow immunochromatographic
approaches.
We report the rapid diagnosis of malaria by aptamer-tethered enzyme capture (APTEC) whereby an aptamer captures biomarker Plasmodium falciparum lactate dehydrogenase (PfLDH) then activity is measured colorimetrically. The robust test was sensitive (limit of detection = 4.9 ng mL(-1)) and could reliably diagnose malaria in clinical blood samples.
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