Highly sensitive detection of microRNA-21 by nitrogen-doped carbon dots-based ratio fluorescent probe via nuclease-assisted rolling circle amplification strategy

Abstract

Highly sensitive and selective detection of microRNA-21 (miRNA-21) in biological samples is critical for the disease diagnosis and cancer treatment. In this study, a nitrogen-doped carbon dots (N-CDs)-based ratio fluorescence sensing strategy was constructed for miRNA-21 detection with high sensitivity and excellent specificity. Bright-blue N-CDs (λex/λem = 378 nm/460 nm) were synthesized by facile one-step microwave-assisted pyrolysis method by using uric acid as the single precursor, and the absolute fluorescence quantum yield and fluorescence lifetime of N-CDs were 35.8% and 5.54 ns separately. The padlock probe hybridized with miRNA-21 firstly and then was cyclized by T4 RNA ligase 2 to form a circular template. At the present of dNTPs and phi29 DNA polymerase, the oligonucleotide sequence in miRNA-21 was prolonged to hybridize with the surplus oligonucleotide sequences in circular template, generating long and reduplicated oligonucleotide sequences containing abundant guanine nucleotides. Separate G-quadruplex sequences were generated after the addition of Nt.BbvCI nicking endonuclease, and then hemin bound with G-quadruplex sequence to construct the G-quadruplex DNAzyme. Such G-quadruplex DNAzyme catalyzed the redox reaction of o-phenylenediamine (OPD) with H2O2, finally producing the yellowish-brown 2,3-diaminophenazine (DAP) (λem = 562 nm). Due to the inner filter effect between N-CDs and DAP, the ratio fluorescence signal of DAP with N-CDs was utilized for sensitive detection of miRNA-21 with detection limit of 0.87 pM. Such approach has practical feasibility and excellent specificity for miRNA-21 analysis during highly homological miRNA family in HeLa cell lysates and human serum samples.