Fluorometric determination of microRNA using arched probe-mediated isothermal exponential amplification combined with DNA-templated silver nanoclusters

Abstract

A highly sensitive fluorometric method is described for the determination of microRNA-141. It is based on the use of arched probe-mediated isothermal exponential amplification reaction (EXPAR) and of DNA-templated silver nanoclusters (DNA-AgNCs). The EXPAR utilizes microRNA-141 as the trigger, polymerases and endonucleases as amplification activators, and two arched probes as exponential amplification templates. This enables the conversion of microRNA to a large number of reporter sequences under isothermal conditions within minutes. The generated reporter sequences act as scaffolds for the synthesis of fluorescent DNA-AgNCs by reduction of Ag (I) with NaBH4. The DNA-AgNCs function as signalling fluorophores with excitation/emission maxima at 540/610 nm. The method exhibits high sensitivity for microRNA-141 with a detection limit as low as 0.87 fM and a dynamic range from 1 fM to 500 fM. The method can distinguish nucleotides in the microRNA-200 family.

Schematic representation of a fluorometric method for sensitive detection of microRNA based on arched probe-mediated isothermal exponential amplification combined with DNA-templated silver nanoclusters.

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Acknowledgments

This work was supported by the Natural Science Foundation of Jiangsu Province (Grant No. BK20171144 and BK20161139) and the Scientific Research Foundation of Jiangsu Provincial Commission of Health and Family Planning, China (Grant No. H2018068).

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Correspondence to Hao Wu or Pei Zou.

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Wu, H., Wu, J., Liu, Y. et al. Fluorometric determination of microRNA using arched probe-mediated isothermal exponential amplification combined with DNA-templated silver nanoclusters. Microchim Acta 186, 715 (2019). https://doi.org/10.1007/s00604-019-3836-4

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Keywords

  • MicroRNA-141
  • Arched probe
  • Silver nanoclusters
  • Isothermal amplification
  • DNA polymerase
  • Nicking endonuclease
  • Human serum