An ultrasensitive electrochemical biosensor for polynucleotide kinase assay based on gold nanoparticle-mediated...

Polynucleotide kinase (PNK) plays an essential role in cellular nucleic acid metabolism and the cellular response to DNA damage. However, conventional methods for PNK assay suffer from low sensitivity and involve multiple steps. Herein, we develop a simply electrochemical method for sensitive detection of PNK activity on the basis of Au nanoparticle (AuNP)-mediated lambda exonuclease cleavage-induced signal amplification. We use [Ru(NH₃)₆]³⁺ as the electrochemically active indicator and design two DNA strands (i.e., strand 1 and strand 2) to sense PNK. The assembly of strand 2 on the AuNP surface leads to the formation of AuNP-strand 2 conjugates which can be subsequently immobilized on the gold electrode through the hybridization of strand 1 with strand 2 for the generation of a high electrochemical signal. The presence of PNK induces the phosphorylation of the strand 2-strand 1 hybrid and the subsequent cleavage of double-stranded DNA (dsDNA) by lambda exonuclease, resulting in the release of AuNP-strand 2 conjugates and [Ru(NH₃)₆]³⁺ from the gold electrode surface and consequently the decrease of electrochemical signal. The PNK activity can be simply monitored by the measurement of [Ru(NH₃)₆]³⁺ peak current signal. This assay is very sensitive with a detection limit of as low as 7.762 × 10^-4UmL^-1 and exhibits a large dynamic range from 0.001 to 10UmL^-1. Moreover, this method can be used to screen the PNK inhibitors, and it shows excellent performance in real sample analysis, thus holding great potential for further applications in biological researches and clinic diagnosis.