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Improving sensitivity of miRNA assay coupled with mismatched catalytic hairpin assembly reaction by optimizing of hairpin annealing conditionsстатья
Статья опубликована в высокорейтинговом журнале
Статья опубликована в журнале из списка Web of Science и/или ScopusДата последнего поиска статьи во внешних источниках: 11 мая 2021 г.
- Авторы: Bodulev Oleg L., Zhao Shulin, Sakharov Ivan Yu
- Журнал: Analytical Chemistry
- Том: 93
- Номер: 17
- Год издания: 2021
- Издательство: American Chemical Society
- Местоположение издательства: United States
- Первая страница: 6824
- Последняя страница: 6830
- DOI: 10.1021/acs.analchem.1c00820
- Аннотация: Mismatched catalytic hairpin assembly (mCHA), a programmable oligonucleotide circuit is one of the promising isothermal amplification methods used in nucleic acid detection. Its limitations are related to a high background noise observed due to the target-independent hybridization of the reacting hairpins (HPs). In this work, it was shown that the introduction of salts such as NaCl and MgCl2 to HP1/HP2 annealing solutions sharply reduces background in mCHA and simultaneously increases signal-to-background (S/B) ratio. Comparison of the salts demonstrated higher activity of MgCl2 compared to NaCl. Similar effect of reducing background was observed with a decrease in the concentration of H1/H2 probes in annealing solutions. Using the favorable annealing conditions allowed the development of ultrasensitive chemiluminescent assay coupled with mCHA for miRNA quantitation. Except mCHA, use of a streptavidin-polyHRP conjugate and enhanced chemiluminescence reaction additionally increased the assay sensitivity. Notably, the optimization of the HP annealing diminished the detection limit of the assay by 2 orders of magnitude and increased the sensitivity and precision of miRNA-141 determination. The discovered fact of reducing background by variation of HP annealing conditions may be valuable not only in mCHA performance, but also likely in other HP-based biochemical methods.
- Добавил в систему: Сахаров Иван Юрьевич