Journal
MICROCHIMICA ACTA
Volume 187, Issue 2, Pages -Publisher
SPRINGER WIEN
DOI: 10.1007/s00604-019-4094-1
Keywords
Fluorescence detection; MicroRNA-21; DNA polymerase; NEase; Hairpin assisted target recycling; Light-up Ag NCs; Strand displacement amplification; Serum analysis
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Funding
- Natural Science Foundation of China [21461002, 21241005, 21562002]
- Key Laboratory of Jiangxi University for Functional Materials Chemistry [FMC15104]
- Jiangxi Provincial Department of Education [GJJ170826, JXJG-14-6]
- Gannan Normal University [16zb19]
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A method is described for the determination of microRNAs via two-stage signal enhancement. This is attained by combining hairpin (HP) assisted cascade isothermal amplification with light-up DNA-Ag nanoclusters. A rationally designed dual-functional HP is used, and microRNA-21 is chosen as a model analyte. At the first stage, upon the hybridization of the microRNA-21 with HP, microRNA recycling via polymerase-displacement reaction and a circulative nicking-replication process are achieved. This generates numerous G-abundant overhang DNA sequences. In the second stage, the above-released G-abundant overhang DNA sequences hybridize with the dark green Ag NCs, and this results in the appearance of bright red fluorescence. Thanks to the two signal enhancement processes, a linear dependence between the fluorescence intensity at 616 nm and the concentration of microRNA-21 is obtained in the range from 1 pM to 20 pM with a detection limit of 0.7 pM. The strategy clearly discriminates between perfectly-matched and mismatched targets. The method was applied to the determination of microRNA-21 in a spiked serum sample.
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