Self-Generation of Distinguishable Fluorescent Probes via a One-Pot Process for Multiple MicroRNA Detection by Liquid Chromatography

To address the challenge of signal production and separation for multiple microRNA (miRNA) detection, in this work, a "one-pot" process to self-generate distinguishable fluorescent probes was developed. Based on a long and short probe amplification strategy, the generated G-quadruplex fluo...

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Bibliographic Details
Published in:Analytical chemistry (Washington) 2023-02, Vol.95 (8), p.4113-4121
Main Authors: Song, Chang, Liu, Chang, Chen, Jisen, Ma, Ziyu, Tang, Sheng, Pan, Ruirong, Suo, Xiaocen, Yan, Zuowei, Lee, Hian Kee, Shen, Wei
Format: Article
Language:English
Subjects:
Analytical chemistry
Automation
Biosensing Techniques - methods
Breast cancer
Chemistry
Chromatography
Chromatography, Liquid
Fluorescein
Fluorescence
Fluorescent Dyes
Fluorescent indicators
G-Quadruplexes
High performance liquid chromatography
Humans
Limit of Detection
Liquid chromatography
MicroRNAs
MicroRNAs - analysis
miRNA
Nanomaterials
Nanotechnology
Nuclease
Nucleic Acid Amplification Techniques - methods
Probes
Ribonucleic acid
RNA
Signal processing
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Summary:To address the challenge of signal production and separation for multiple microRNA (miRNA) detection, in this work, a "one-pot" process to self-generate distinguishable fluorescent probes was developed. Based on a long and short probe amplification strategy, the generated G-quadruplex fluorescent dye-free probes can be separated and detected by a high-performance liquid chromatography-fluorescence platform. The free hairpin probes enriched in guanine with different lengths and base sequences were designed and could be opened by the target miRNAs (miRNA-10b, miRNA-21, and miRNA-210). Cleaved G-quadruplex probes with fluorescent signal could be generated in a one-pot process after a duplex-specific nuclease-based cleavage, and the detection of multiple miRNAs could be realized in one run. No solid nanomaterials were applied in the assay, which avoided the blocking of the column. Moreover, without modification of expensive fluorescein, the experimental cost was greatly reduced. The one-pot reaction process also eliminated tedious preparation steps and suggested feasibility of automation. The limits of detection of miRNA-10b, miRNA-21, and miRNA-210 were 2.19, 2.20, and 2.75 fM, respectively. Notably, this method was successfully applied to multiplex detection of miRNAs in serum samples from breast cancer patients within 30 min.
ISSN:0003-2700
1520-6882
DOI:10.1021/acs.analchem.2c04941