Sypabekova, Marzhan2024-08-132024-08-132019Sypabekova, M. (2019). Impedance Based Aptasensor For The Detection Of Mycobacterium Tuberculosis Secreted Protein Mpt64. Nazarbayev University School of Engineering and Digital Scienceshttps://nur.nu.edu.kz/handle/123456789/8199Tuberculosis (TB) detection remains a significant healthcare issue in the developing world owing to a number of challenges. Current diagnostics are based on microbiological culturing, sputum smear microscopy, and nucleic acid amplification tests. These methods suffer from limitations such as batch to batch variations, frequent contaminations, low sensitivity, and the requirement for special facilities, expensive devises, reagents, and trained personnel. This thesis describes the development of the sensitive oligonucleotide-based aptasensor for the detection of TB biomarker MPT64 protein. The dissertation investigates the selection and use of ssDNA aptamers to detect MPT64 using the electrochemical impedance spectroscopy (EIS). Aptamers serve as bio-recognition elements in this study, and they have numerous advantages including cheap cost, ease of modification and long shelf life. The combination of aptamers with the EIS offers sensitive detection since the change in EIS signal can be recorded as the result of analyte binding event based not only on molecular interaction level but also on electron transfer levels. As the result 17 unique aptamer sequences were purified and analyzed. One aptamer with dissociation equilibrium constant KD of 8.92 nM was selected and the surface chemistry was optimized based on ssDNA aptamer modified with a long linker and 6-mercaptohexanol as a co-adsorbent at 1/100 ratio. The selected aptamer was further immobilized on an interdigitated microelectrode and connected to a portable potentiostat. The detection time for aptasensor was found to be 15 min. The aptasensor was tested on clinical samples and showed increased binding to TB (+) samples as compared to TB (-) samples. The integration of the aptasensor with the in house built fluidic chamber and liquid flow rate within chamber was also investigated. The work in this thesis is significant as it can contribute to the diagnosis of TB (non-invasive), monitoring of anti-TB treatment in infected people and hence to socio-economic development of the country. It is the first portable aptasensor which is developed using aptamers and EIS as a detection technique that can provide fast clinical sample analysis (reduced from 3 h to 15 min) as well as elimination of using of extra reagents, equipment, and personnel.enAttribution-NonCommercial-ShareAlike 3.0 United StatesType of access: EmbargoPhD thesis