Development of a bio-nanoparticle DNA-based biosensor for tuberculosis detection using isothermal amplification

Tuberculosis (TB) is the world's second deadliest infectious disease and it has been identified as a leading cause of death among HIV-positive patients. The standard test for TB diagnosis is smear sputum microscopy, which is not able to identify half of the positive TB infections. Several molecular based techniques have been explored for the rapid detection of Mycobacterium tuberculosis, but they require highly specialized equipment, which makes them expensive and difficult to implement in resource-constrained peripheral laboratories. The main objective of the dissertation was to develop a portable, bio-nanoparticle DNA based biosensor to detect M. tuberculosis using dextrin coated gold nanoparticles as electrochemical labels and isothermal helicase-dependent thermophilic amplification (tHDA). The gold nanoparticles were synthesized using a novel alkaline, dextrin driven alternative to the citrate-coated gold nanoparticles. Particles were obtained with a diameter between 5.9 and 16.8 nm and the effects of dextrin concentration, temperature, and pH over diameter and production rate were evaluated. The dextrin coated gold nanoparticles were functionalized with DNA probes and used as electrochemical labels for the DNA biosensor. The tHDA reaction was successfully optimized to amplify a specific fragment of M. tuberculosis (from the IS6110 gene) in a regular heating block. Therefore, a thermocycler is not required for the DNA amplification, like in PCR. The obtained detection limit of the DNA biosensor was 0.01ng/&mgr;l of isothermally amplified DNA fragments using synthetic targets.