An electrochemical biosensor for the activity assay of polynucleotide kinase and inhibitor screening

Abstract

A sensitive and selective electrochemical biosensor was fabricated for polynucleotide kinase (PNK) activity assay and inhibitor screening based on phos-tag-biotin mediated double signal amplification, where streptavidin and alkaline phosphatase-conjugated biotin (biotin-ALP) were used as the signal amplification units. After the immobilization of probe DNA on the electrode surface, it was hybridized with complementary DNA containing the 5′-OH terminal, the OH can be phosphorylated under the catalytic effect of PNK in the presence of ATP. The generated phosphate group at the 5′-terminal can be further recognized by phos-tag-biotin, where phos-tag-biotin was not only used as the specific recognition reagent for the phosphate group, but also the capture reagent for streptavidin to induce the further assembly of biotin-ALP. Under the catalytic effect of ALP, the substrate of p-nitrophenyl phosphate disodium salt hexahydrate (PNPP) can be hydrolyzed to produce p-nitrophenol (PNP). According to the relationship between the electrochemical response of PNP and the logarithmic value of PNK concentration, a linear range of 0.01–5 units per mL and a low detection limit of 0.0027 unit per mL were achieved. The developed method showed high selectivity. The inhibition effect of (NH₄)₂SO₄ and Na₂HPO₄ was also evaluated and the IC₅₀ values were calculated.