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

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.