Detection of T4 polynucleotide kinase activity with immobilization of TiO2 nanotubes and amplification of Au nanoparticles

Determination of nucleotide kinase activity is valuable due to its importance in regulating nucleic acid metabolism. Herein, we describe a strategy for simply and accurately determining nucleotide kinase activity by TiO2 nanotubes mediated signal transition and Au nanoparticles amplification. In this method, DNA containing 5′-hydroxyl group is self-assembled onto a gold electrode and used as a substrate for T4 polynucleotide kinase (PNK). By the specific immobilization affinity of TiO2 nanotubes with the phosphorylated DNA, TiO2 nanotubes were linked with phosphorylated substrate DNA on the electrode. And then Au nanoparticles modified 5′-phosphate DNA was conjugated with the TiO2 nanotubes and hybridized with methylene blue labeled signal DNA. Because gold nanoparticles have high loading of signal indicator methylene blue, the electrochemical signal is generated and amplified. It presents an excellent performance with wide linear range and low detection limit. Additionally, inhibition effects of some salts have also been investigated. The developed method is a potentially useful tool in researching the interactions between proteins and nucleic acids and provides a diversified platform for a kinase activity assay.

► TiO2 nanotubes for the immobilization of phosphorylated DNA were synthesized by a novel method.
► T4 polynucleotide kinase (PNK) activity was determined in the sandwich DNA structure.
► Au nanoparticles were used for signal amplification.
► Wide linear range and low detection limit were obtained in the present system.