Ratio fluorescence analysis of T4 polynucleotide kinase activity based on the formation of graphene quantum dots-copper nanoclusters nanohybrid

In this work, a ratio fluorescence method was developed for T4 polynucleotide kinase (PNK) activity analysis based on the formation of dual-emitting graphene quantum dots-copper nanoclusters (GQDs-CuNCs) nanohybrid. Amino capped single-strand DNA (ssDNA) was firstly used to modify GQDs (GQDs-ssDNA) and then hybridize with its complementary DNA strand to form double-strands DNA functionalized GQDs (GQDs-dsDNA), dsDNA of GQDs-dsDNA can act as the effective template for preparation CuNCs with fluorescence emission at 594 nm. When dsDNA of GQDs-dsDNA was phosphorylated through T4 PNK and subsequently degraded via λ exonuclease (λ exo) to produce mononucleotides and GQDs-ssDNA, the formation of fluorescence CuNCs in GQDs-CuNCs was blocked due to the lack of effective dsDNA substrate, during which the fluorescence of GQDs at 446 nm in nanohybrid was nearly not influenced. Thus, with the CuNCs serving as the reporter and GQDs as the reference signal, T4 PNK activity can be monitored through the change of fluorescence intensity ratio (F594/F446) in the range of 0.01-10 U mL-1 with detection limit (LOD) 0.0037 U mL-1. Furthermore, the practicality of this T4 PNK activity analysis strategy in complex sample was performed in cell lysates.