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Colorimetric and visual determination of microRNA via cycling signal amplification using T7 exonuclease

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Abstract

A sensitive and specific bioassay based on cyclic enzymatic amplification was developed for determination of microRNA (miRNA) by taking advantage of the exodeoxyribonuclease activity of T7 exonuclease (T7 Exo). In the presence of miRNA, DNA/RNA duplexes are formed by hybridization of miRNA and capture probes (Cp). Then, the Cp is digested to release miRNA into the next amplification cycle assisted by T7 Exo. This leads to the digestion of numerous Cp molecules. The broken Cp does no longer hybridize with hairpin probes (Hp) to unveil G-quadruplex DNAzyme (GDNAs). However, in the absence of miRNA, the Hp hybridizes with Cp to unveil GDNAs. The generated GDNAs form assemblies with hemin to form the G-quadruplex/hemin DNAzyme complex which is capable of catalyzing the oxidation of the substrate ABTS by H2O2. Upon cyclic enzymatic amplification, the output signal is reduced accordingly, this resulting in a “signal-off” signal best acquired at a wavelength of 418 nm. The lower detection limit is 0.69 pM (at an S/N ratio of 3). The assay involves efficient signal amplification, is homogeneous and isothermal, and enables visual detection. It provides a simple, rapid and sensitive platform for use in clinical diagnostics.

Schematic of a method for colorimetric and visual detection of miRNA using T7 exonuclease-based cycling signal amplification.

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Acknowledgements

This work was supported by the National Natural Science Foundation of China (81572080, 81101638), Natural Science Foundation Project of Chongqing (cstc2014kjrc-qnrc10001), and Achievement Transfer Project of Institutions of Higher Education in Chongqing (KJZH14205).

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Authors and Affiliations

  1. Key Laboratory of Clinical Laboratory Diagnostics (Ministry of Education), College of Laboratory Medicine, Chongqing Medical University, Chongqing, 400016, China

    Ye Sang, Yongjie Xu, Xinmin Li, Jiangling Wu & Shijia Ding

  2. Department of Laboratory Medicine, Guizhou Provincial People’s Hospital, Guiyang, 550002, China

    Yongjie Xu

  3. The Center for Clinical Molecular Medical Detection, The First Affiliated Hospital of Chongqing Medical University, Chongqing, 400016, China

    Lulu Xu & Wei Cheng

Authors
  1. Ye Sang
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  2. Yongjie Xu
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  3. Lulu Xu
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  4. Wei Cheng
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  5. Xinmin Li
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  6. Jiangling Wu
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  7. Shijia Ding
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Correspondence to Shijia Ding.

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The authors declare that they have no conflict of interest. This article does not contain any studies with animals performed by any of the authors.

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Ye Sang and Yongjie Xu are contributed equally to this work.

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Sang, Y., Xu, Y., Xu, L. et al. Colorimetric and visual determination of microRNA via cycling signal amplification using T7 exonuclease. Microchim Acta 184, 2465–2471 (2017). https://doi.org/10.1007/s00604-017-2238-8

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  • DOI: https://doi.org/10.1007/s00604-017-2238-8

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