An enzyme-free and label-free electrochemical biosensor for kinase

Polynucleotide kinase (PNK) plays a significant role in the cell signaling pathway and the metabolism damage of cellular nucleic acid. Therefore, it is greatly pivotal to detect the PNK activity and its inhibition. In this work, an enzyme-free and label-free electrochemical sensor for detecting the PNK activity is developed using a glassy carbon electrode (GCE) modified with gold nanoparticles (AuNPs) and zirconia (ZrO2) based on the PNK cata-lyzing the phosphorylation of the 5 '-hydroxyl group of DNA. In the presence of PNK, the DNA was transformed to phosphorylated DNA (pDNA) which preferentially bound with ZrO2/AuNPs/GCE over the unphosphorylated DNA by virtue of the strong affinity between ZrO2 and the phosphate groups. Once the phosphorylated DNA was captured by the ZrO2/AuNPs/GCE, methylene blue (MB), an electroactive indicator, was loaded and produced a characteristic cathodic peak current. The cathodic peak current exhibited a linear correlation to the logarithm of PNK concentration ranging from 0.01 U/mL to 10 U/mL. Moreover, this method can be used to screen the PNK inhibitors and shows excellent performance in real sample analysis, thus holding great potential for further application biological researches and clinic diagnosis.