Lateral flow nucleic acid biosensor for sensitive detection of microRNAs based on the dual amplification strategy of duplex-specific nuclease and hybridization chain reaction

Na Ying; Chuanjing Ju; Xiuwei Sun; Letian Li; Hongbiao Chang; Guangping Song; Zhongyi Li; Jiayu Wan; Enyong Dai

doi:10.1371/journal.pone.0185091

Lateral flow nucleic acid biosensor for sensitive detection of microRNAs based on the dual amplification strategy of duplex-specific nuclease and hybridization chain reaction

PLoS One. 2017 Sep 25;12(9):e0185091. doi: 10.1371/journal.pone.0185091. eCollection 2017.

Authors

Na Ying^{1

2}, Chuanjing Ju^{1

3

4}, Xiuwei Sun^{1

5}, Letian Li¹, Hongbiao Chang^{1

6}, Guangping Song^{1

7}, Zhongyi Li¹, Jiayu Wan¹, Enyong Dai⁸

Affiliations

¹ Institute of Military Veterinary, Academy of Military Medical Sciences, Changchun, China.
² East China Sea Fisheries Research Institute, China Academy of Fishery Sciences, Shanghai, China.
³ The General Hospital of FAW, Changchun, China.
⁴ The Fourth Hospital of Jilin University, Changchun, China.
⁵ College of Animal Science and Technology, Jilin Agricultural University, Changchun, China.
⁶ School of Life Science and Technology, Changchun University of Science and Technology, Changchun, China.
⁷ Heilongjiang Bayi Agricultural University, Daqing, China.
⁸ Department of Oncology and Hematology, China-Japan Union Hospital of Jilin University, Changchun, China.

Abstract

MicroRNAs (miRNAs) constitute novel biomarkers for various diseases. Accurate and quantitative analysis of miRNA expression is critical for biomedical research and clinical theranostics. In this study, a method was developed for sensitive and specific detection of miRNAs via dual signal amplification based on duplex specific nuclease (DSN) and hybridization chain reaction (HCR). A reporter probe (RP), comprising recognition sequence (3' end modified with biotin) for a target miRNA of miR-21 and capture sequence (5' end modified with Fam) for HCR product, was designed and synthesized. HCR was initiated by partial sequence of initiator probe (IP), the other part of which can hybridize with capture sequence of RP, and was assembled by hairpin probes modified with biotin (H1-bio and H2-bio). A miR-21 triggered cyclical DSN cleavage of RP, which was immobilized to a streptavidin (SA) coated magnetic bead (MB). The released Fam labeled capture sequence then hybridized with the HCR product to generate a detectable dsDNA. This polymer was then dropped on lateral flow strip and positive result was observed. The proposed method allowed quantitative sequence-specific detection of miR-21 (with a detection limit of 2.1 fM, S/N = 3) in a dynamic range from 100 fM to 100 pM, with an excellent ability to discriminate differences in miRNAs. The method showed acceptable testing recoveries for the determination of miRNAs in serum.

MeSH terms

Biosensing Techniques / methods*
Endonucleases
Humans
MicroRNAs / analysis*
MicroRNAs / blood
MicroRNAs / genetics*
Nucleic Acid Amplification Techniques / methods*
Nucleic Acid Heteroduplexes
Nucleic Acid Hybridization / methods

Substances

MIRN21 microRNA, human
MicroRNAs
Nucleic Acid Heteroduplexes
Endonucleases

Grants and funding

This study was funded by The Basic Work of Science and Technology Special of China (NO.2013FY113600), National Key Technologies R&D Program of China (No.2013BAD12B04), Science and technology development plans of Jilin Province (20150101105JC) and The National Key Research and Development Program of China (No.2016YFD0501000). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.