Exonuclease III-assisted strand displacement reaction-driven cyclic generation of G-quadruplex strategy for homogeneous fluorescent detection of melamine

Development of flexible, sensitive, selective and simple melamine detection is in high demand and of great significance. Here, we proposed a homogeneous turn-off mode fluorescent strategy to detect melamine by coupling the process of exonuclease III-assisted (Exo III) amplification with the strand displacement reaction (SDR)-driven assembly of DNA G-quadruplex structures. Melamine (M) could bind to the thymine (T) base through hydrogen bonding to form a T-M-T structure, which inhibits the subsequent nucleic acid amplification reaction and formation of the G-quadruplex structure. DNA strand (G1) served as both a recognition probe and a signal probe, thus greatly simplifying the experimental design and operation. Furthermore, to improve the detection sensitivity, an ingeniously designed segment of double-stranded DNA (dsDNA, P1-G2) that contained the G-quadruplex sequence (G2) and a partially hybridized signal trigger strand (P1) was added to the system. Following the SDR and Exo III-assisted amplification, G1-DNA and G2-DNA were released and the cyclic production of G-quadruplex structures was initiated. The generated DNA G-quadruplex bound to the NMM (a fluorescent dye, N-methyl mesoporphyrin IX), providing enhanced fluorescence signals. This allowed for the highly sensitive detection of melamine at concentrations as low as 25 fM. The proposed fluorescent detection method of melamine concentration displayed a good linear relationship ranging from 100 fM to 100 pM, which showed improved performance over that of the non-enzyme-assisted sensing system (limit of detection is 15 pM). Additionally, this method exhibited a high selectivity towards the target molecule, melamine, in comparison to other substances. Furthermore, the method was used for the assay of melamine in real milk samples and satisfactory experiment results were obtained. The novel strategy developed in this research may be considered as a potential route for highly sensitive, simple, selective and accurate assay of small molecules.