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Label-free detection of microRNA: two-stage signal enhancement with hairpin assisted cascade isothermal amplification and light-up DNA-silver nanoclusters

Abstract

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.

Schematic representation of microRNA detection by integrating hairpin assisted cascade isothermal amplification with light-up DNA Ag nanoclusters. With microRNA, G-abundant overhang DNA sequences from amplification reaction hybridize with dark green Ag nanoclusters to produce a concentration-dependent bright red fluorescence

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Acknowledgments

This work was supported by the Natural Science Foundation of China (21461002, 21241005 and 21562002), Key Laboratory of Jiangxi University for Functional Materials Chemistry (FMC15104), Jiangxi Provincial Department of Education (GJJ170826 and JXJG-14-6), and Gannan Normal University (16zb19).

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Correspondence to YongRong Xie or QingYou Cai.

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Li, M., Xu, X., Zhou, Z. et al. Label-free detection of microRNA: two-stage signal enhancement with hairpin assisted cascade isothermal amplification and light-up DNA-silver nanoclusters. Microchim Acta 187, 141 (2020). https://doi.org/10.1007/s00604-019-4094-1

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Keywords

  • Fluorescence detection
  • MicroRNA-21
  • DNA polymerase
  • NEase
  • Hairpin assisted target recycling
  • Light-up Ag NCs
  • Strand displacement amplification
  • Serum analysis