Unlocking the hidden talent of DNA: Unexpected catalytic activity for colorimetric assay of alkaline phosphatase

Carboxylic acids have been efficiently used to activate H₂O₂ to form even more potent oxidant-peroxy acids through enzyme-catalyzed processes. By employing acetic acid as the activator, herein we report for the first time that cofactor-free DNA displays unexpected activity in H₂O₂-mediated oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) under mild conditions. A series of 10-nt oligonucleotides were rationally designed with various combinations of double nucleotides including TG, AG, CG, TA and AC respectively, which demonstrates that the catalytic performance of DNA is highly dependent upon the sequence composition, strand length and continuous nucleotides. Inspired by phosphate-induced inhibition effects on the formation of peracetic acid, an ultrasensitive assay was well-established for monitoring alkaline phosphatase (ALP) on the basis of double terminal-phosphorylated G-rich oligonucleotides. Phosphorylated DNA not only serves as the substrate for ALP-catalyzed hydrolysis, but also acts as the enzyme-like catalyst for signal amplification. Quantitative determination of ALP is realized in a linear range from 0.05 to 15 mU/mL, resulting in the limit of detection of 0.01 mU/mL. The rapid and reliable test also has great potential in analyzing serum samples for practical disease diagnosis.