A ratiometric fluorescent sensor for sensitive detection of UDG using poly(thymine)-templated copper nanoclusters and DAPI with exonuclease III assisted amplification

As one of critical base excision repair enzymes, uracil-DNA glycosylase (UDG) can specifically repair uracil-induced DNA damage to maintain the genome integrity of organisms. Moreover, abnormal expression of UDG is related to various cancers and other serious diseases. Therefore, it is essential to accurately and sensitively monitor UDG activity. In this work, a ratiometric fluorescence method using poly-(thymine) DNA template copper nanoclusters (Cu NCs) and 4′,6-diamidino-2-phenylindole (DAPI) as the output signals is developed for simple, selective, and sensitive detection of UDG. A double-stranded DNA (dsDNA) consisting of a uracil-containing single chain and a trigger sequence is designed as a substrate dsDNA. Meanwhile, another dsDNA comprising 30 pairs of A-T bases ((AT)30 dsDNA) is designed to be partially complementary to the trigger chain. The (AT)30 dsDNA itself has no blunt or recessed 3′ end and cannot be cut by exonuclease III (Exo III). With the Exo III-assisted amplification, adding UDG results in the success synthesis of red-emissive Cu NCs, while the dsDNAs in the system are hydrolyzed and the fluorescence signal of DAPI is weak. In contrast, without addition of UDG, the fluorescence intensity of Cu NCs is low, while the DAPI emits significantly enhanced fluorescence signal. The proposed method presents a detection limit of 5.0 × 10−5 U mL−1 and can be applied to the HeLa cell lysate sample with satisfactory results. Additionally, the kinetic parameter is studied and the UDG inhibition effect is evaluated. In summary, this work provides a potential platform for DNA repair enzyme-related analysis and clinical diagnosis.