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Browsing Articles by Subject "aptamer"

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    ENZYME LINKED OLIGONUCLEOTIDE ASSAY FOR THE SENSITIVE DETECTION OF SARS-COV-2 VARIANTS
    (Frontiers in Cellular and Infection Microbiology, 2022-09-29) David, Michael Shola; Kanayeva, Damira
    The exponential spread of COVID-19 has prompted the need to develop a simple and sensitive diagnostic tool. Aptamer-based detection assays like ELONA are promising since they are inexpensive and sensitive. Aptamers have advantages over antibodies in wide modification, small size, in vitro selection, and stability under stringent conditions, which aid in scalable and reliable detection. In this work, we used aptamers against SARS-CoV-2 RBD S protein to design a simple and sensitive ELONA detection tool. Screening CoV2-RBD-1C and CoV2-RBD-4C aptamers and optimizing assay conditions led to the development of a direct ELONA that can detect SARS-CoV-2 RBD S glycoprotein in buffer solution and 0.1 % human nasal fluid with a detection limit of 2.16 ng/mL and 1.02 ng/mL, respectively. We detected inactivated Alpha, Wuhan, and Delta variants of SARS-CoV-2 with the detection limit of 3.73, 5.72, and 6.02 TCID50/mL, respectively. Using the two aptamers as capture and reporter elements, we designed a more sensitive sandwich assay to identify the three SARS-CoV-2 variants employed in this research. As predicted, a lower detection limit was obtained. Sandwich assay LOD was 2.31 TCID50/mL for Alpha, 1.15 TCID50/mL for Wuhan, and 2.96 TCID50/mL for Delta. The sensitivity of sandwich ELONA was validated using Alpha and Wuhan variants spiked in 0.1% human nasal fluid sample condition and were detected in 1.41 and 1.79 TCID50/ mL LOD, respectively. SEM was used to visualize the presence of viral particles in the Delta variant sample. The effective detection of SARS-CoV-2 in this study confirms the potential of our aptamer-based technique as a screening tool.
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    A NOVEL SSDNA APTAMER TARGETING CARCINOEMBRYONIC ANTIGEN: SELECTION AND CHARACTERIZATION
    (Biology, 2022-10-20) Yunussova, Nigara; Sypabekova, Marzhan; Zhumabekova, Zhazira; Matkarimov, Bakhyt; Kanayeva, Damira
    One of the major causes of a drastically shorter life expectancy and one of the most prevalent diseases in the world today is cancer. Given the data on the rise in cancer cases throughout the world, it is obvious that, despite the diagnostic techniques currently being used, there is a pressing need to develop precise and sensitive techniques for early diagnosis of the disease. A high degree of affinity and specificity towards particular targets is maintained by the short nucleic acid molecules known as aptamers. Aptamers outperform antibodies due to their unique benefits, such as their simplicity in synthesis and modification, lack of toxicity, and long-term stability. Utilizing an accurate recognition element and a robust signal transduction mechanism, molecular diagnostics can be extremely sensitive and specific. In this study, development of new single-stranded DNA aptamers against CEA for use in cancer diagnostics was accomplished using SELEX and NGS methods. As a result of 12 iterative SELEX rounds, nine aptamer candidates against CEA were developed. NGS comparative analysis revealed that round twelve had an enriched number of aptamers that were specifically bound, as opposed to round eight. Among the selected nine sequences characterized by bioinformatics analysis and ELONA, an aptamer sequence with the highest specificity and affinity for the target protein was identified and further examined. Aptamer sequence (6) was screened in a concentration-dependent assay, specificity analysis was performed, and its potential secondary and tertiary structures were predicted, which enabled us to test one of the possible putative interactions with CEA. Finally, aptamer sequence (6) labelled with a Cy5 fluorescent tag was used in confocal microscopy to observe its binding towards the CEA expressed in HT-29 human colon adenocarcinoma cell line
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    SENSITIVE DETECTION OF SARS-COV-2 VARIANTS USING AN ELECTROCHEMICAL IMPEDANCE SPECTROSCOPY BASED APTASENSOR
    (International Journal of Molecular Sciences, 2022) Kurmangali, Assem; Dukenbayev, Kanat; Kanayeva, Damira
    The global pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARSCoV- 2) caused a threat to public health and a worldwide crisis. This raised the need for quick, effective, and sensitive detection tools to prevent the rapid transmission rate of the infection. Therefore, this study aimed to develop an electrochemical impedance spectroscopy (EIS)-based aptasensor employing an interdigitated gold electrode (IDE) to detect SARS-CoV-2 Spike (S) glycoprotein and viral particles. This allowed us to sensitively detect SARS-CoV-2 S glycoprotein with a limit of detection (LOD) of 0.4 pg/mL in a buffer solution and to obtain a linear increase for concentrations between 0.2 to 0.8 pg/mL with high specificity. The proposed aptasensor also showed a good sensitivity towards the heat-inactivated SARS-CoV-2 variants in a buffer solution, where the Delta, Wuhan, and Alpha variants were captured at a viral titer of 6.45 0.16 103 TCID50/mL, 6.20 104 TCID50/mL, and 5.32 0.13 102 TCID50/mL, respectively. Furthermore, the detection of SARS-CoV-2 performed in a spiked human nasal fluid provided an LOD of 6.45 0.16 103 TCID50/mL for the Delta variant in a 50 L sample and a detection time of less than 25 min. Atomic force microscopy images complemented the EIS results in this study, revealing that the surface roughness of the IDE after each modification step increased, which indicates that the target was successfully captured. This label-free EIS-based aptasensor has promising potential for the rapid detection of SARS-CoV-2 in complex clinical samples.