The development of a novel DNAzyme-based biosensor for the detection of nucleic acid targets applicable for point of care diagnostics

Rapid, sensitive and affordable molecular diagnostic tests that can operate in point of care (POC) settings are in high demand, but their availability and use in the field remains limited. A rapid diagnosis is imperative for effective and timely treatment of disease, ultimately leading to improved patient outcomes. In this thesis, novel strategies for enhancing the detection of nucleic acid targets were developed, providing new avenues for POC disease diagnosis. This was achieved by integrating nucleic acid detection technologies, namely PlexZymes™, with isothermal signal amplification. Universal signal amplification was demonstrated through the development of a novel class of molecules referred to as Subzymes. These versatile molecules contain two components which enable it to function as both a catalytic nucleic acid enzyme and a nucleic acid substrate. Subzymes were shown to be valuable tools for the construction of DNAzyme-based nucleic acid biosensing systems. Here, it was established that some varieties of Subzymes can be inhibited by attaching them to the surfaces of micro-particles. Moreover, their catalytic activity can be subsequently restored upon their release from the surfaces. These molecules are named ‘Cleavage-Activated-Subzymes’ (CASzymes) and were coupled with PlexZymes™ for the enhanced detection of nucleic acid targets. Cleavage of a CASzyme’s substrate results in the release and activation of the surface-bound DNAzyme. These DNAzymes were shown to initiate feedback cascades by cleaving other CASzymes and activating additional DNAzymes. The active DNAzymes were shown to be capable of cleaving reporter substrates to generate signal which ultimately resulted in improved target sensitivities and faster rates of detection. These signal amplification methods demonstrate rapid detection (< 30 minutes) of specific nucleic acid targets, with a detection limit of 1 fM, without the use of protein-enzymes. The Subzyme cascades are constructed from low-cost materials and operate under isothermal reaction conditions, negating the need for expensive and sophisticated equipment. These features are important for the development of POC diagnostic tests, especially where access to medical facilities is limited. Thus, Subzymes enable rapid and affordable detection of nucleic acid targets and demonstrate a strong potential for POC applications in the future