Electrochemical detection of T4 polynucleotide kinase activity based on magnetic Fe₃O₄@TiO₂ nanoparticles triggered by a rolling circle amplification strategy

An ultrasensitive electrochemical detection of the activity and inhibition of T4 polynucleotide kinase (T4 PNK) was developed by using magnetic Fe₃O₄@TiO₂ core-shell nanoparticles, which was triggered by a rolling circle amplification strategy (Fe₃O₄@TiO₂-RCA). We used Fe₃O₄@TiO₂ as a substrate to anchor a DNA primer. DNA S1 with 5'-OH termini was phosphorylated in the presence of T4 PNK and ATP, which was adsorbed on the surface of Fe₃O₄@TiO₂ NPs and served as the primer for subsequent RCA reactions. After adding circular template DNA S2, RCA was initiated in the presence of phi29 DNA polymerase and dNTPs. Then, Fc-labeled DNA S3 (Fc-S3) was hybridized with RCA. The obtained Fe₃O₄@TiO₂-RCA was adsorbed on the surface of a magnetic gold electrode (MGE) by magnetic enrichment, resulting in an enhanced electrochemical signal. The T4 PNK activity can be monitored by measuring the electrochemical signal generated. This electrochemical assay is sensitive to the activity of T4 PNK with a dynamic linear range of 0.00001-20 U/mL and a low detection limit of 3.0 × 10^-6 U/mL. The proposed strategy can be used to screen the T4 PNK inhibitors, so it has great potential in the discovery of nucleotide kinase-target drug and early clinical diagnosis of cancer.