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 λ 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 λ 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 λ 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 U⋅L−1 limit of detection. The strategy also provides the basis for a screening platform for inhibitors of ALP.