Sensitive Nanochannel Biosensor for T4 Polynucleotide Kinase Activity and Inhibition Detection
Sensitive Nanochannel Biosensor for T4 Polynucleotide Kinase Activity and Inhibition Detection
复制标题
用于 T4 多核苷酸激酶活性和抑制检测的灵敏纳米通道生物传感器
DOI：
10.1021/ac302875p
复制
发表时间：
2013-01-01
影响因子：
7.4
通讯作者：
Li, Jinghong
中科院分区：
文献类型：
--
作者：
Lin, Lei;Liu, Yang;Li, Jinghong
5'-多核苷酸激酶是催化5'-羟基末端核酸磷酸化的一类重要酶。这个过程调节许多重要的细胞事件，尤其是链损伤和中断期间的 DNA 修复。核苷酸激酶的活性和抑制已被证明对细胞核酸调节和代谢有明显的影响。在这里，我们描述了一种新型纳米通道生物传感器，用于监测 T4 多核苷酸激酶 (PNK) 的活性和抑制，PNK 是 5'-激酶家族的著名成员，在细胞对 DNA 损伤的反应中发挥着重要作用。基于功能化纳米通道系统和偶联的λ核酸外切酶裂解反应，纳米通道传感平台对激酶分析表现出高灵敏度和便利性。生物素标记的 dsDNA 通过在通道内形成紧密排列的 DNA 结构，有效阻断链霉亲和素修饰的纳米通道。当 dsDNA 被 PNK 磷酸化，然后立即被 lambda 核酸外切酶切割时，孔堵塞作用几乎消失。这种 PNK 引起的微观结构清晰度可以通过纳米通道系统直接、准确地监测，这得益于其对有效孔径变化的高灵敏度。此外，改装的便利性和机械的坚固性也保证了测试平台的稳定性。该策略在 PNK 活性分析和抑制评估中均表现出出色的分析性能。这种简单而灵敏的纳米通道生物传感器在开发用于基础生化过程研究、分子靶向治疗和临床诊断的片上高通量检测方面显示出巨大潜力。
5'-Polynucleotide kinase is a crucial class of enzyme that catalyzes the phosphorylation of nucleic acids with 5'-hydroxyl termini. This process regulates many important cellular events, especially DNA repair during strand damage and interruption. The activity and inhibition of nucleotide kinase have proven to be an evident effect on cellular nucleic acid regulation and metabolism. Here, we describe a novel nanochannel biosensor for monitoring the activity and inhibition of T4 polynucleotide kinase (PNK), a famous member of the 5'-kinase family playing a major role in the cellular responses to DNA damage. On the basis of the, functionalized nanochannel system and coupled lambda exonuclease cleavage reaction, the nanochannel-sensing platform exhibits high sensitivity and convenience toward kinase analysis. Biotin-labeled dsDNA effectively blocks the streptavidin-modified nanochannel through forming a closely packed arrangement of DNA structure inside the channel. When dsDNA is phosphorylated by PNK and then immediately cleaved by lambda exonuclease, the pore-blocking effect almost disappears. This PNK-induced microstructural distinctness can be directly and accurately monitored by the nanochannel system, which benefits from its high sensitivity to the change of the effective pore size. Furthermore, modification convenience and mechanical robustness also ensure the stability of the test platform. This as-proposed strategy exhibits excellent analytical performance in both PNK activity analysis and inhibition evaluation. The simple and sensitive nanochannel biosensor shows great potential in developing on-chip, high-throughput assays for fundamental biochemical process research, molecular-target therapies, and clinic diagnostics.