Poly(thymine)-Templated Selective Formation of Copper Nanoparticles for Alkaline Phosphatase Analysis Aided by Alkyne–Azide Cycloaddition “Click” Reaction

Alkaline phosphatase (ALP) is closely associated with many health disorders like liver diseases and bone diseases, which is a routine index of blood examination. In the current study, a fluorescent method for the detection of ALP is established based on poly(thymine)-templated selective formation of copper nanoparticles (CuNPs) and alkyne–azide cycloaddition “click” reaction. Generally, in the presence of ALP, l-ascorbic acid-2-phosphate is hydrolyzed, and the generated ascorbic acid reduces Cu2+ to Cu+, which further catalyzes alkyne–azide cycloaddition click reaction between two poly(thymine) segments. The two DNA fragments (18 thymine) are thus ligated, forming the DNA template (36 thymine), which is effective for the synthesis of CuNPs. Experimental results show that the fluorescence response of CuNPs increases with higher ALP concentration from 0.1 to 40 U/mL, and the limit of detection is as low as 0.05 U/mL. Furthermore, the proposed method is highly selective and is capable of detecting ALP in biological fluidlike serum, which suggests its wide biomedical applications.