Ratiometric fluorescence sensor based on carbon dots as internal reference signal and T7 exonuclease-assisted signal amplification strategy for microRNA-21 detection

• Published in: Analytica chimica acta Vol. 1103; pp. 212 - 219
• Main Authors: Wang, Zhenzhen, Xue, Zhiqiang, Hao, Xiaoli, Miao, Chenfang, Zhang, Jianzhong, Zheng, Yanjie, Zheng, Zongfu, Lin, Xinhua, Weng, Shaohuang
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 22-03-2020
• Subjects: Bacteriophage T7 - enzymology; Biosensing Techniques - methods; Carbon - chemistry; Carbon dots; DNA Probes - chemistry; DNA Probes - genetics; DNA, Single-Stranded - chemistry; DNA, Single-Stranded - genetics; Exodeoxyribonucleases - chemistry; Fluorescent Dyes - chemistry; Gastrointestinal cancer; Gastrointestinal Neoplasms - blood; Humans; Limit of Detection; MicroRNAs - blood; MicroRNAs - genetics; miRNA-21; Nucleic Acid Amplification Techniques - methods; Nucleic Acid Hybridization; Quantum Dots - chemistry; Ratiometric fluorescence sensor; Reproducibility of Results; Spectrometry, Fluorescence; T7 exonuclease-assisted signal amplification strategy; T7 exonuclease-assisted signal amplification strategy; Carbon dots; Ratiometric fluorescence sensor; Gastrointestinal cancer; miRNA-21
• ISSN: 0003-2670; 1873-4324
• DOI: 10.1016/j.aca.2019.12.068

The expression level of miRNA-21 is closely related to the occurrence and development of cancer, especially in gastrointestinal cancer. Monitoring miRNA-21 has clinical application in the diagnosis and evaluation of gastrointestinal cancer. A turn-on ratiometric fluorescence bioassay based on the T7 exonuclease-mediated cyclic enzymatic amplification method was developed for miRNA-21 determination by using carbon dots (CDs) and FAM-labeled ssDNA as the signal source. CDs demonstrated the triple functions of built-in internal fluorescence, probe carrier, and quencher in this strategy. In the absence of miRNA-21, FAM-labeled ssDNA would be adsorbed and quenched by CDs. The addition of miRNA-21 induced cycle hydrolysis from the 5′ end by the T7 exonuclease and then released the short-cleaved FAM-labeled oligonucleotides. Then, the increased FAM signal (FFAM) and the stable CD signal (FCDs) would be tested through a ratiometric routine for the quantification of miRNA-21. The FFAM/FCDs value showed a good linear relationship with the concentration of miRNA-21 in the range of 0.05–10 nM, and the detection limit for miRNA-21 was 1 pM with excellent selectivity and reproducibility. Furthermore, this sensor successfully evaluated the expression level of miRNA-21 in clinical blood samples from healthy individuals and gastrointestinal cancer patients, and the results were highly consistent with those of qRT-PCR, suggesting the great clinical application value in the diagnosis of cancer associated with miRNA-21 expression levels.
[Display omitted]
•A turn-on ratiometric fluorescence bioassay based on the T7 exonuclease and carbon dots (CDs) was developed for miRNA-21.•CDs demonstrated the triple functions of built-in internal fluorescence, probe carrier, and quencher in this strategy.•The assay showed a good linear relationship with low detection limit, excellent selectivity and reproducibility for miRNA-21.•The expression level of miRNA-21 is closely related to the occurrence and development of gastrointestinal cancer.•This method successfully evaluated miRNA-21 in clinical blood samples in accord with the results from qRT-PCR.