Ultrasensitive fluorescent detection of nucleic acids based on label-free enzymatic-assisted cascade signal amplification.

A simple and homogenous label-free fluorescent method based on target-triggered and enzymatic-assisted isothermal cascade signal amplification was established for nucleic acid detection. This amplification method consists of two circuits that depend on polymerase and nicking endonuclease. In the presence of a target, a hairpin probe was opened to initiate the polymerization and nicking reaction, producing target analogues and G-rich sequences, and releasing the original target DNA. The released target and produced target analogues then trigger a new amplification cycle. Large amounts of G-rich sequences were generated through this cascade amplification process. The fluorogenic dye thioflavin T (ThT) specifically recognized G-rich sequences to form a G-quadruplex/ThT complex, which induced a strong fluorescence intensity. The approach was ultrasensitive for nucleic acid detection, and the detection limit was as low as 5.6 fM. The system discriminates single-nucleotide mutations in DNA and provides a promising strategy for nucleic acid detection in complex biological samples. This simple, label-free and ultrasensitive approach is a promising tool for biomedical research and clinical diagnostics.