Issue 67, 2018, Issue in Progress

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

Abstract

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.

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

Supplementary files

Article information

Article type
Paper
Submitted
18 Sep 2018
Accepted
29 Oct 2018
First published
14 Nov 2018
This article is Open Access
Creative Commons BY-NC license

RSC Adv., 2018,8, 38436-38444

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

Y. Zhang, X. Fang, Z. Zhu, Y. Lai, C. Xu, P. Pang, H. Wang, C. Yang, C. J. Barrow and W. Yang, RSC Adv., 2018, 8, 38436 DOI: 10.1039/C8RA07745B

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