An electrochemical assay of polynucleotide kinase activity based on streptavidin–gold nanoparticles and enzymatic amplification

An electrochemical method for the determination of polynucleotide kinase (PNK) activity has been proposed with a dual signal amplification strategy. The method relies on the phosphorylation-induced DNA digestion and two-step signal amplification using streptavidin–gold nanoparticle biocomplexes and alkaline phosphatase. In the presence of T4 PNK, the 5′-terminus of the immobilized PNK substrate probe was phosphorylated, and the substrate probe would be cleaved by λ exonuclease after being hybridized with the complementary detection probe biotinylated in the 3′-hydroxyl terminus. As a result, the detection probe would be released, with an electrochemical signal decrease, due to less biotinylated alkaline phosphatase loading on the electrode surface. The electrochemical signal exhibited a linear correlation to the logarithm of PNK concentration ranging from 0.01 U mL−1 to 5 U mL−1 with the detection limit of 0.01 U mL−1. The inhibiting effect of (NH4)2SO4 on the activity of PNK was also evaluated.