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Microchimica Acta

, Volume 183, Issue 11, pp 3061–3067 | Cite as

Electrochemical strategy for ultrasensitive detection of microRNA based on MNAzyme-mediated rolling circle amplification on a gold electrode

  • Jianru Yang
  • Min Tang
  • Wei Diao
  • Wenbin Cheng
  • Ye Zhang
  • Yurong Yan
Original Paper

Abstract

The authors describe an electrochemical strategy for ultrasensitive and specific detection of microRNA (miRNA). It is based on both multicomponent nucleic acid enzyme (MNAzyme) amplification and rolling circle amplification (RCA). In the presence of target miRNAs, partial enzyme A (partzyme A) and partial enzyme B (partzyme B) are assembled to form active MNAzymes. Once formed, the MNAzymes catalyze the cleavage of the hairpin substrates to liberate biotinylated fragments which hybridized with the capture probes immobilized on a gold electrode. The RCA is then initiated to form a product that binds many detection probes. Finally, the amperometric signal (best acquired at a working voltage of 0.22 V vs. Ag/AgCl) is obtained by employing the streptavidinylated alkaline phosphatase as the enzyme. This biosensor has a 1.66 fM detection limit, and a dynamic range that extends from 10 fM to 1 nM. It displays specificity down to single mismatch discrimination of target miRNAs and good reproducibility. It was successfully applied to the determination of miRNA in total RNA samples extracted from human breast adenocarcinoma MCF-7 cells.

Graphical abstract

Cascade signal amplification strategy for microRNA electrochemical detection based on multicomponent nucleic acid enzyme (MNAzyme)-mediated rolling circle amplification. MCH: 6-mercapto-1-hexanol; BSA: bovine serum albumin; dNTP: deoxy-ribonucleoside triphosphate; ST-AP: streptavidin-alkaline phosphatase; α-NP: α-naphthyl phosphate

Keywords

Electrochemical biosensor Electrochemical impedance spectroscopy Differential pulse voltammetry Square wave voltammetry Alkaline phosphatase Streptavidin human breast adenocarcinoma MCF-7 cells DNA polymerase T4 DNA ligase 

Notes

Acknowledgments

This work was funded by the National Natural Science Foundation of China (81101638) and the Science and Technology Plan Project of Yu Zhong District of Chongqing (20150114).

Compliance with ethical standards

The author(s) declare that they have no competing interests.

Supplementary material

604_2016_1958_MOESM1_ESM.doc (339 kb)
ESM 1 (DOC 339 kb)

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Copyright information

© Springer-Verlag Wien 2016

Authors and Affiliations

  1. 1.Key Laboratory of Clinical Laboratory Diagnostics (Ministry of Education), College of Laboratory MedicineChongqing Medical UniversityChongqingChina

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