A turn-on fluorescent method for determination of the activity of alkaline phosphatase based on dsDNA-templated copper nanoparticles and exonuclease based amplification

The authors describe a method for the determination of the activity of alkaline phosphatase (ALP) that utilizes dsDNA-templated copper nanoparticles (CuNPs) coupled to enzymatic amplification via lambda exonuclease. A hybrid of a DNA modified with a phosphate moiety at the 5'-end (P-DNA) and a P-DNA complementary sequence (cP-DNA) is employed as the dsDNA substrate for ALP. In the absence of ALP, the dsDNA is cleaved by the lambda exonuclease, which hinders the formation of CuNPs which display fluorescence with excitation/emission peaks at 340/565 nm. However, ALP-mediated hydrolysis of the 5'-phosphoryl end impedes the cleavage of dsDNA by the lambda exonuclease, and this promotes the formation of fluorescent dsDNA-templated CuNPs via ascorbate-mediated reduction. Under the optimized experimental conditions, this method exhibits a high specificity to ALP and has a 0.1 Uai...L-1 limit of detection. The strategy also provides the basis for a screening platform for inhibitors of ALP.