Enzymatic polymerization-based formation of fluorescent copper nanoparticles for the nuclease assay

Abstract In this work, inspired by the idea that the DNA fragments of nuclease cleavage exactly can serve as the primers for terminal deoxynucleotidyl transferase (TdT) mediated polymerization, a versatile and sensitive method for nuclease assay was developed based on the enzymatic polymerization and poly(thymine)-templated copper nanoparticles (CuNPs). Specifically, in the presence of target nuclease, the DNA substrate was firstly degraded as mononucleotides and oligonucleotide fragments. Employing the fragments as primers, the TdT-assisted polymerization was initiated to generate poly(thymine) templates on which fluorescent CuNPs could be synthesized. The fluorescence intensity of CuNPs was detected for the nuclease assay. So different nucleases assay could be realized just by changing the designs of DNA substrates of the corresponding nucleases. For the versatility demonstration, two nucleases, Exo III and EcoR V as the model of exonuclease and endonuclease, respectively, were detected with this method. It was proved that the proposed method was of high selectivity and sensitivity, and the limits of detections of Exo III and EcoR V were found to be 0.0138 and 0.0115 U/mL, respectively. What’s more, the satisfactory result of EcoR V detection in human serum indicates that this method is capable to detect nuclease in real sample.