Publications by authors named "I Yu Sakharov"
Article Synopsis
- A new assay was developed to measure the activity of the nicking endonuclease (NE) Nt.Bst9I using biotinylated DNA oligonucleotides as a substrate.
- To enhance the assay's sensitivity, a chemiluminescent detection system was implemented, utilizing a streptavidin-polyperoxidase conjugate and an enhanced chemiluminescence reaction.
- The assay methods were designed for microtiter plates, facilitating automated analysis with ELISA equipment, and the heterogeneous format proved to be more sensitive than the homogeneous-heterogeneous format.
A heterogeneous sensitive microRNA-155 assay based on a new isothermal amplification method, called catalytic hairpin assembly with oligonucleotide release (CHAOR), was developed. The principle of CHAOR was studied by non-denaturing electrophoresis. To detect the amplification product, a polyperoxidase-streptavidin conjugate (molar ratio 1:80) and an enhanced chemiluminescence reaction were used, which made it possible to increase assay sensitivity.
View Article and Find Full Text PDFMessenger ribonucleic acids (mRNAs) comprise a class of small nucleic acids carrying genetic information, which exhibit very important role in medical research and diagnosis. If only the mean mRNA expression levels of the mRNA population are considered in medical research, important information linking mRNA expression and cellular function may be lost. Single-cell analysis provides valuable insights into studying its heterogeneity, signaling, and stochastic gene expression.
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- Two types of assays (heterogeneous and homogeneous-heterogeneous) were developed to quantify hsa-miR-141-3p using isothermal circular strand-displacement polymerization (ICSDPR) and enhanced chemiluminescence for improved sensitivity.
- The heterogeneous assay proved to be more sensitive than the homogeneous-heterogeneous assay, with detection limits of 51 fM and 10 pM, respectively, and an amplification index of 100.
- The assays successfully determined the levels of miRNA-141 in various human cell lines, revealing distinct copy numbers per cell (Caco-2: 3400, HepG2: 1400, MCF-7: 1300, HeLa: 470).
In the present work, we describe the development of a chemiluminescent enzyme-linked oligonucleotide assay coupled with mismatched catalytic hairpin assembly (mCHA) amplification for the quantitative determination of microRNA-155. To improve its sensitivity, a polymerase-free mCHA reaction was applied as an isothermal amplification method. The detection limit of the proposed assay was 400 fM.
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