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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.

rsc advances

A sensitive electrochemical assay for T4 polynucleotide kinase activity based on titanium dioxide nanotubes and a rolling circle amplification strategy