Theranostics 2019; 9(1):279-289. doi:10.7150/thno.28474

Research Paper

An innovative “unlocked mechanism” by a double key avenue for one-pot detection of microRNA-21 and microRNA-141

Weipan Peng1*, Qian Zhao1*, Minghui Chen1, Jiafang Piao1, Weichen Gao1, Xiaoqun Gong1,2✉, Jin Chang1✉

1. School of Life Sciences, Tianjin University and Tianjin Engineering Center of Micro-Nano Biomaterials and Detection-Treatment Technology (Tianjin), Tianjin 300072, China
2. State Key Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin 300072, China
*Authors who contributed equally to this work.

This is an open access article distributed under the terms of the Creative Commons Attribution (CC BY-NC) license ( See for full terms and conditions.
Peng W, Zhao Q, Chen M, Piao J, Gao W, Gong X, Chang J. An innovative “unlocked mechanism” by a double key avenue for one-pot detection of microRNA-21 and microRNA-141. Theranostics 2019; 9(1):279-289. doi:10.7150/thno.28474. Available from

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The accurate and quantitative detection of microRNAs (miRNAs) as next-generation, reliable biomarkers will provide vital information for cancer research and treatment. However, their unique, intrinsic features pose quite a challenge for miRNA profiling, especially for multiplexed detection. Thus, there is a strong and an ever-growing need to develop an accurate, simple, sensitive and specific miRNA sensing method.

Methods: In this study, a simple and novel sensor is presented that uses a flow cytometry (FCM) method based on the double key “unlocked mechanism” and a fluorescence enrichment signal amplification strategy. The “unlocked mechanism” was cleverly designed via using hairpin DNA probes (HDs) labeled by fluorescent particles (FS) as the lock to block part of them, which can specifically hybridize with the probe on polystyrene microparticles (PS). The target miRNA and duplex-specific nuclease (DSN) forming the double key can specifically open the HDs and cleave a single-stranded DNA (ssDNA) into DNA/RNA dimers circularly in order to unlock the special part of the HDs to be specially enriched further on the PS.

Results: The designed sensor with a hairpin structure and DSN special performance was found to have a high specificity. The circularly unlocking fluorescent probes and fluorescent signal enrichment can be beneficial for achieving a high sensitivity with a detection limit of 3.39 fM for miRNA-21. Meanwhile, the performance of multiplexing was estimated by simultaneous detection of miR-21 and miR-141, and the method also allowed for miR-21 detection in breast cancer blood samples.

Conclusion: The designed sensor based on an “unlocked mechanism” and a signal enrichment strategy resulted in a one-pot, highly specific and sensitive detection of multiplex miRNAs. The whole detection without the need for a complex purification process is based on a FCM and is expected to have a great value in cancer diagnosis and biomedical research.

Keywords: miRNA, “unlocked mechanism”, DSN, signal enrichment