Synthesis of gold nanoparticles for rapid genotyping of M. tuberculosis using rolling circle amplification and nanoflare technology

Abstract: Tuberculosis (TB) is an airborne disease caused by Mycobacterium tuberculosis, with an incidence in a quarter of the world population. Despite the scientific and technological advances, an effective diagnostic method has not yet been found that allows an early diagnosis and, also, to detect the strain present in the patient. The combination of nanotechnology with molecular diagnostics has shown promising advances offering new possibilities, such as the development of nanoflares.  Nanoflares represent a new class of molecular probes, composed of gold nanoparticles functionalized with a recognition sequence that can be amplified by rolling circle amplification (RCA) technique, producing a fluorescence signal.  This thesis focuses in the synthesis of gold nanoparticles, with different coatings and sizes, as well as their subsequent application in the preparation and optimization of nanoflares for the genotyping of synthetic M. tuberculosis targets using RCA technique. The different preparations of nanoflares have an impact in the assay sensitivity, showing two times increase in sensitivity for citrate-coated nanoparticles with respect to those coated with PEG. Furthermore, it was observed that the sensitivity is directly related to the synthesized particle size.  Sensitivity is also affected by the application of a purification post-treatment of the synthesis product. This post-treatment reduces the sensitivity of nanoflares by up to 37% but, by contrast, extends its useful life.  The results obtained are shown as a proof of concept for a future cost-effective, rapid and robust in situ diagnostic method that identifies the strain of tuberculosis present in the patient.