Abstract
The global Tuberculosis (TB) pandemic remains a major cause of mortality; it killed 1.3 million people in 2020, second only to COVID-19. Patients with active TB are highly infectious, so a rapid and reliable diagnosis is essential to initiate the isolation, treatment and contact tracing protocols required to control this disease.
Poorly resourced communities rely on direct sputum smear microscopy as a primary TB diagnostic. This technique requires skilled personnel to assess multiple samples from each patient, and typically detects only 70\% of active cases. A rapid Point of Care (POC) test is urgently required for use by health workers with limited training. This must be accurate, robust, and affordable.
These requirements are not met by a nucleic acid amplification or antibody test. While the latter has been used successfully for COVID-19 screening, they are unsuitable in areas of high TB prevalence: Around one-third of the world's population have been exposed to TB and carry antibodies, but most are not infectious.
Mycolic Acids (MAs), present in sputum, are excellent biomarkers for rapid and accurate TB diagnosis. They are a unique component of Mycobacterial cell walls, and secreted in large amounts by the tuberculosis bacilli to promote disease processes. Current MA detection protocols are confined to very well equipped laboratories, and typically include extraction and chemical derivitisation, followed by chromatography and/or Mass Spectroscopy (MS).
This thesis aims to develop a molecular detection system, suitable for use in a rapid POC diagnostic. The proposed method may be likened to Thermal Desorption Spectroscopy (TDS): Sample components are differentiated on the basis of the temperature at which they desorb from a crystalline surface, heated in a programmed cycle. This technique leverages the strong dependence of the binding enthalpies of aliphatic molecules on their chain length.
Traditional TDS is performed in an ultra-high vacuum, with adsorbate sublimation monitored via MS. The proposed Thermal Sorption Spectroscopy (TSS) system monitors desorption (and adsorption) in an aqueous environment. MS is replaced by a simple measurement of interfacial capacitance, a technique employed by existing affinity biosensors. A key advantage of the proposed approach is that highly specific surface-bound probe molecules, used in existing biosensors, are not needed.
This thesis describes the design, construction and testing of a prototype TSS system. It incorporates a thermocycled three-electrode fluid cell; and a capacitance measurement system, consisting of a potentiostat and a lock in amplifier. The TSS hardware incorporates new Intellectual Property (IP): An ultra-fast thermocycler; and a novel quadrature sampling lock-in amplifier design.
Proof of concept for TSS has been demonstrated by the detection of several exemplar molecules: Adenine, MAs, and various androgens. Lipids such as MA proved difficult to solubilise; protocols incorporating surfactants were trialled, but require further development work. The thermocycler was also used as a stand-alone system to successfully perform a DNA amplification on a sample retained within a paper cartridge, demonstrating potential for its use in a Polymerase Chain Reaction (PCR) based diagnostic system. A paper cartridge could incorporate printed electrodes for the non-optical detection of PCR products.