An emerging class of molecules known as �aptamers� offer new, exquisitely sensitive ways of detecting molecular signatures associated with different disease states. These synthetic single- stranded nucleic acid molecules bind any target with high affinity and specificity. Aptamers have been widely used in therapeutics and diagnostics, but are hitherto unapplied to tuberculosis (TB), a disease which, in South Africa, is the single biggest killer of individuals infected with HIV. We therefore seek to develop an aptamer-based electrochemical biosensor for the detection of an established biomarker associated with extra-pulmonary TB (EPTB). We used an iterative process known as the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) to generation single-stranded DNA aptamers. Further characterization was performed using an Enzyme-Linked Oligonucleotide Assay (ELONA) and surface plasmon resonance (SPR). Bioinformatic tools were used to assess the stability; secondary structure predictions, and to perform truncations of the selected aptamer. At 60% enrichment, aptamers were cloned and sequenced. Six aptamers with the highest binding affinities were selected for further characterization, and most could detect the target protein at concentrations below 500 ng/50�l (10 ng/�l). The lowest limit of detection for the aptamers was found to be 18.75 nM, where aptamers A1 and B2 bound significantly (p<0.05). The binding kinetics showed that aptamers bound with dissociation constants (KD) in the picomolar range, with the lowest being for aptamers H12 and A1, which was found to be 3.86E-11 and 9.22E-11, repectively. Aptamers appear to be a promising technology for the diagnosis of TB. This area is believed to have the potential to circumvent all the shortcomings that current tests have. Further work needs to focus on assessing their diagnostic accuracy on a larger scale (including examining the impact of HIV co-infection) and miniaturizing the technology so it is suitable for use at the point-of-care.

Characterization of aptamer-based probes and the development of an electrochemical biosensor: Potential TB diagnostic tools

248th National Meeting (2014)

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