Label-free and sensitive fluorescent sensing of ten-eleven translocation enzyme via cascaded recycling signal amplifications.

The sensitive detection of ten-eleven translocation (TET) dioxygenase is of significance for understanding the demethylation mechanism of 5-methylocytosine (5mC), which is responsible for a wide range of biological functions that can affect gene expression in eukaryotic species. Here, a non-label and sensitive fluorescence biosensing method for TET assay using TET1 as the model target molecule is established on the basis of target-triggered Mg-dependent DNAzyme and catalytic hairpin assembly (CHA)-mediated multiple signal amplification cascades. 5mC sites in the hairpin DNA probe are first oxidized by TET1 into 5-carboxycytosine, which are further reduced by pyridine borane into dihydrouracil, followed by its recognition and cleavage by the USER enzyme to liberate active DNAzyme and G-quadruplex sequences from the probe. The DNAzyme further cyclically cleaves the substrate hairpins to trigger subsequent CHA reaction and DNAzyme cleavage cycles for yielding many G-quadruplex strands. Thioflavin T dye then intercalates into G-quadruplexes to cause a magnificent increase of fluorescence for high sensitivity assay of TET1 with 47 fM detection limit. And, application of this method for TET1 monitoring in diluted serum has also been confirmed.