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An “off-on” fluorescent switch assay for microRNA using nonenzymatic ligation-rolling circle amplification

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Abstract

The authors describe a fluorometric switch assay for microRNA (miRNA). It is based on nonenzymatic ligation-rolling circle amplification (NELRCA). A click chemistry-mediated functionalized linear template was prepared by self-cycling. It is capable of forming a modified circular template (MLT) with a triazole linkage for amplified agent. MiRNA can recognize a binding region of the MLT, activates the NELRCA, and converts into rolling circle amplification products (RCPs). Subsequently, the added FAM-labeled signal probes (FAM-SPs) can hybridize to the RCPs. This RCPs/FAM-SPs duplex is less adsorbed onto the graphene oxide (GO) so that fluorescence (best measured at excitation/emission wavelengths of 490/520 nm) increases. In contrast to classical GO-based adsorption and displacement sensing system, the designed hybridization and adsorption fluorescent switch-on platform can reduce the assay time, and improve the detection efficiency of target. Under optimal conditions, the sensing platform has a detection limit as low as 0.75 fM. It can well discriminate target miRNA from other kinds of miRNA. Conceivably, this method can be extended to the trace detection of other nucleic acid biomarkers, and simultaneous recognition of multiple targets, thereby improving the early clinical diagnostic accuracy of cancer.

A hybridization and adsorption switch-on sensing system was fabricated on nonenzymatic ligation-rolling circle amplification. Modified circular template with 1,4-connect triazole linkage was adopted as an identified and amplified agent for high sensitive detection of microRNA.

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Acknowledgements

This work was financially supported by the Natural Science Research Foundation of China (81672112, 81171415) and the Chongqing Yuzhong District Science and Technology Project (20140108).

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Authors and Affiliations

  1. Key Laboratory of Laboratory Medical Diagnostics, Ministry of Education, Department of Laboratory Medicine, Chongqing Medical University, Chongqing, 400016, People’s Republic of China

    Yi Li, Qinli Pu, Junlong Li, Lili Zhou, Yiyi Tao, Wen Yu & Guoming Xie

  2. Clinical Laboratories, The First Affiliated Hospital of Chongqing Medical University, Chongqing, 400016, People’s Republic of China

    Yuxia Li

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  1. Yi Li
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  2. Qinli Pu
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  3. Junlong Li
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  4. Lili Zhou
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  7. Wen Yu
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Correspondence to Guoming Xie.

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Li, Y., Pu, Q., Li, J. et al. An “off-on” fluorescent switch assay for microRNA using nonenzymatic ligation-rolling circle amplification. Microchim Acta 184, 4323–4330 (2017). https://doi.org/10.1007/s00604-017-2475-x

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  • DOI: https://doi.org/10.1007/s00604-017-2475-x

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