Browsing by Author "Sypabekova, Marzhan"

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    IMPEDANCE BASED APTASENSOR FOR THE DETECTION OF MYCOBACTERIUM TUBERCULOSIS SECRETED PROTEIN MPT64
    (Nazarbayev University School of Engineering and Digital Sciences, 2019) Sypabekova, Marzhan
    Tuberculosis (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.
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    Role of viruses in the development of breast cancer
    (Infectious Agents and Cancer, 2013-09-02) Alibek, Kenneth; Kakpenova, Ainur; Mussabekova, Assel; Sypabekova, Marzhan; Karatayeva, Nargis
    The most common cancer worldwide among women is breast cancer. The initiation, promotion, and progression of this cancer result from both internal and external factors. The International Agency for Research on Cancer stated that 18-20% of cancers are linked to infection, and the list of definite, probable, and possible carcinogenic agents is growing each year. Among them, biological carcinogens play a significant role. In this review, data covering infection-associated breast and lung cancers are discussed and presented as possible involvements as pathogens in cancer. Because carcinogenesis is a multistep process with several contributing factors, we evaluated to what extent infection is significant, and concluded that members of the herpesvirus, polyomavirus, papillomavirus, and retrovirus families definitely associate with breast cancer. Detailed studies of viral mechanisms support this conclusion, but have presented problems with experimental settings. It is apparent that more effort needs to be devoted to assessing the role of these viruses in carcinogenesis, by characterizing additional confounding and synergistic effects of carcinogenic factors. We propose that preventing and treating infections may possibly stop or even eliminate certain types of cancers.
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    Selection, characterization, and application of DNA aptamers for detection of Mycobacterium tuberculosis secreted protein MPT64
    (Tuberculosis, 2017-05-01) Sypabekova, Marzhan; Bekmurzayeva, Aliya; Wang, Ronghui; Li, Yanbin; Nogues, Claude; Kanayeva, Damira; Marzhan, Sypabekova
    Abstract Rapid detection of Mycobacterium tuberculosis (Mtb), an etiological agent of tuberculosis (TB), is important for global control of this disease. Aptamers have emerged as a potential rival for antibodies in therapeutics, diagnostics and biosensing due to their inherent characteristics. The aim of the current study was to select and characterize single-stranded DNA aptamers against MPT64 protein, one of the predominant secreted proteins of Mtb pathogen. Aptamers specific to MPT64 protein were selected in vitro using systematic evolution of ligands through exponential enrichment (SELEX) method. The selection was started with a pool of ssDNA library with randomized 40-nucleotide region. A total of 10 cycles were performed and seventeen aptamers with unique sequences were identified by sequencing. Dot Blot analysis was performed to monitor the SELEX process and to conduct the preliminary tests on the affinity and specificity of aptamers. Enzyme linked oligonucleotide assay (ELONA) showed that most of the aptamers were specific to the MPT64 protein with a linear correlation of R2 = 0.94 for the most selective. Using Surface Plasmon Resonance (SPR), dissociation equilibrium constant KD of 8.92 nM was obtained. Bioinformatics analysis of the most specific aptamers revealed the existence of a conserved as well as distinct sequences and possible binding site on MPT64. The specificity was determined by testing non-target ESAT-6 and CFP-10. Negligible cross-reactivity confirmed the high specificity of the selected aptamer. The selected aptamer was further tested on clinical sputum samples using ELONA and had sensitivity and specificity of 91.3% and 90%, respectively. Microscopy, culture positivity and nucleotide amplification methods were used as reference standards. The aptamers studied could be further used for the development of medical diagnostic tools and detection assays for Mtb.
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    Tuberculosis diagnosis using immunodominant, secreted antigens of Mycobacterium tuberculosis
    (Tuberculosis, 2013-07-01) Bekmurzayeva, Aliya; Sypabekova, Marzhan; Kanayeva, Damira; Aliya, Bekmurzayeva
    Summary Tuberculosis (TB) remains a major public health concern in most low-income countries. Hence, rapid and sensitive TB diagnostics play an important role in detecting and preventing the disease. In addition to established diagnostic methods, several new approaches have been reported. Some techniques are simple but time-consuming, while others require complex instrumentation. One prominent and readily available approach is to detect proteins that Mycobacterium tuberculosis secretes, such as Mpt64, the 6-kDa early secreted antigenic target (Esat6), the 10-kDa culture filtrate protein (Cfp10), and the antigen 85 (Ag85) complex. Although their functions are not fully understood, a growing body of molecular evidence implicates them in M. tuberculosis virulence. Currently these biomarkers are either being used or investigated for use in skin patch tests, biosensor analyses, and immunochromatographic, immunohistochemical, polymerase chain reaction-based, and enzyme-linked immunosorbent assays. This review provides a comprehensive discussion of the roles these immunodominant antigens play in M. tuberculosis pathogenesis and compares diagnostic methods based on the detection of these proteins with more established tests for TB.