A widely applicably method is described for fluorometric determination of targets such as microRNA and viral DNA. It is making use of a Mg(II)-dependent DNAzyme and a G-quadruplex. In the absence of analyte, an inactive DNAzyme is formed by the hybridization of split DNAzymes and substrate. On addition of target analyte, the end of each strand of the split DNAzymes bind the analyte. This leads to the generation of an active DNAzyme. In the presence of Mg(II), the activated DNAzyme is formed and can cleave the substrate strand. Hence, the caged G-quadruplex sequences will be released. These released G-quadruplexes combine with thioflavin T to generate a G-quadruplex/thioflavin T complex and thereby cause amplified fluorescence. The method shows a 70 pM detection limit for H5N1 and works over a wide linear range 1 nM to 400 nM. Conceivably, this detection scheme has a wide scope in that it may be applied to other assays for microRNAs and DNAs by variation of the type of DNAzyme. Graphical abstract Schematic presentation of target detection: the DNAzyme cannot cleave the substrate strand when target is absent. Once the target is added, the active DNAzyme can cleave the substrate strand in the presence of Mg2+, resulting in significant fluorescence enhancement when the release of the caged G-quadruplex sequences binding with 2-[4-(dimethylamino)phenyl]-3,6-dimethylbenzothiazolium chloride (ThT).
Keywords: DNAzyme; Fluorescence; G-quadruplexes; Multiple detection.