A sensitive electrochemical assay for T4 polynucleotide kinase activity based on titanium dioxide nanotubes and a...

An ultrasensitive electrochemical biosensor was developed for detection of T4 polynucleotide kinase (T4 PNK) activity based on titanium dioxide nanotubes (TiO₂ NTs) and a rolling circle amplification (RCA) strategy. In this study, the immobilized T4 PNK substrate probe with a 5' terminus hydroxyl was phosphorylated by T4 PNK in the presence of adenosine triphosphate (ATP), and the resulting 5-phosphoryl can be linked with the TiO₂ NTs and further conjugated with the phosphate-labeled primer. RCA was initiated by adding circular template, phi29 DNA polymerase and deoxyribonucleoside 5-triphosphate mixture (dNTPs). Biotin-labeled probes are chosen as a signal indicator by strong biotin-streptavidin interaction and the high loading of horseradish peroxidase-streptavidin (HRP-SA) for electrochemical signal generation and amplification. A dual-signaling amplification strategy has been established, which exhibited an excellent performance with a wide linear range from 0.0001-15 U mL^-1 and a low detection limit of 0.00003 U mL^-1 for T4 PNK detection. The inhibition effect of (NH₄)₂SO₄ on the activity of T4 PNK is also evaluated. This new dual-signaling electrochemical biosensor can be used for the detection of the activity and inhibition of other nucleic acid enzymes.