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首页> 外文期刊>RSC Advances >A sensitive electrochemical assay for T4 polynucleotide kinase activity based on titanium dioxide nanotubes and a rolling circle amplification strategy
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A sensitive electrochemical assay for T4 polynucleotide kinase activity based on titanium dioxide nanotubes and a rolling circle amplification strategy

机译：基于二氧化钛纳米管的T4多核苷酸激酶活性的敏感电化学测定和滚环扩增策略

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摘要

An ultrasensitive electrochemical biosensor was developed for detection of T4 polynucleotide kinase (T4 PNK) activity based on titanium dioxide nanotubes (TiO _(2) 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 TiO _(2) 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 (NH _(4) ) _(2) SO _(4) 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.
机译：开发了一种超灵敏的电化学生物传感器，用于检测基于二氧化钛纳米管（TiO_（2）NTs）的T4多核苷酸激酶（T4 PNK）活性和滚环扩增（RCA）策略。在这项研究中，在三磷酸腺苷（ATP）存在的情况下，固定的具有5'末端羟基的T4 PNK底物探针被T4 PNK磷酸化，所得的5-磷酰基可与TiO_（2）NTs连接，并进一步连接与磷酸盐标记的引物偶联。通过添加环状模板，phi29 DNA聚合酶和脱氧核糖核苷5-三磷酸混合物（dNTP）来启动RCA。生物素-链霉亲和素的强相互作用和高负荷辣根过氧化物酶-链霉亲和素（HRP-SA）的选择将生物素标记的探针用作信号指示剂，用于电化学信号的产生和放大。已经建立了双信号放大策略，该策略具有出色的性能，线性范围为0.0001–15 U mL〜（？1），T4 PNK检测的检测下限为0.00003 U mL〜（？1）。还评估了（NH _（4）_（2）SO _（4）对T4 PNK活性的抑制作用。这种新型的双信号电化学生物传感器可用于检测活性和抑制其他核酸酶。

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