Construction of a Quencher-Free Cascade Amplification System for Highly Specific and Sensitive Detection of Serum Circulating miRNAs

Circulating miRNAs are a newly emerging class of noninvasive biomarkers, and the accurate quantification of their expression is essential to the biological research and early clinic diagnosis. Herein, we demonstrate the construction of a quencher-free cascade amplification system for highly sensitiv...

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Bibliographic Details
Published in:Analytical chemistry (Washington) 2020-06, Vol.92 (12), p.8546-8552
Main Authors: Wang, Li-Juan, Ren, Ming, Wang, Hou-Xiu, Qiu, Jian-Ge, Jiang, BingHua, Zhang, Chun-Yang
Format: Article
Language:English
Subjects:
2-Aminopurine
Amplification
Binding
Biological research
Biomarkers
Cell Line, Tumor
Chemistry
Construction
Diagnosis
Exonuclease
Fluorescence
Humans
Lung cancer
Lung carcinoma
MicroRNAs - blood
MicroRNAs - genetics
miRNA
Non-small cell lung carcinoma
Nucleic Acid Amplification Techniques
Probes
Real-Time Polymerase Chain Reaction
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Summary:Circulating miRNAs are a newly emerging class of noninvasive biomarkers, and the accurate quantification of their expression is essential to the biological research and early clinic diagnosis. Herein, we demonstrate the construction of a quencher-free cascade amplification system for highly sensitive detection of serum circulating miRNAs. The target miRNA can hybridize with the linear probe to induce the cyclic strand displacement amplification (SDA) (cycle I) for the production of the binding probes. The binding probe can subsequently react with the 2-aminopurine (2-AP)-hairpin probe to induce the recycling exonuclease cleavage of 2-AP-hairpin probes (cycle II), releasing the triggers and 2-AP molecules simultaneously. The released trigger can hybridize with the free linear probe to start new cycles I and II amplifications. Through multiple rounds of cascade amplifications, a large number of 2-AP molecules are released, generating an enhanced fluorescence signal. This method exhibits a large dynamic range of 8 orders of magnitude and a detection limit of 0.16 aM. It can differentiate a single-base mismatch in miR-486-5p, quantify miR-486-5p in lung cancer cells at various stages, and even discriminate the expressions of serum circulating miR-486-5p in healthy persons from that in nonsmall-cell lung carcinoma (NSCLC) patients. Moreover, this assay can be rapidly carried out in one step under isothermal condition in a label-free manner, holding promising applications in point-of-care diagnosis and prognosis of lung cancers.
ISSN:0003-2700
1520-6882
DOI:10.1021/acs.analchem.0c01385