College of Chemistry, Chemical Engineering and Materials Science, Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in Universities of Shandong, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Shandong Provincial Key Laboratory of Clean Production of Fine Chemicals, Shandong Normal University, Jinan, 250014, PR China.
College of Chemistry, Chemical Engineering and Materials Science, Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in Universities of Shandong, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Shandong Provincial Key Laboratory of Clean Production of Fine Chemicals, Shandong Normal University, Jinan, 250014, PR China.
Biosens Bioelectron. 2020 Feb 15;150:111865. doi: 10.1016/j.bios.2019.111865. Epub 2019 Nov 11.
We develop a low-background electrochemical biosensor for one-step detection of uracil DNA glycosylase (UDG) based on the host-guest interaction and iron-embedded nitrogen-rich carbon nanotube (Fe-N-C) that mimics enzyme-mediated electrocatalysis to achieve signal amplification. In this work, Fe-N-C is initially immobilized on a glassy carbon electrode, followed by the immobilization of β-cyclodextrin (β-CD). We construct the signal probes by assembling the methylene blue (MB)-labeled hairpin DNAs onto the surface of Au nanoparticles (AuNPs) to form the MB-hairpin/AuNP probes. Due to the steric effect of AuNPs and the stem-loop structure of hairpin DNA, MB is prevented from entering the cavity of β-CD on the electrode. In contrast, UDG enables the removal of uracil from the U•A pairs in the stem of hairpin DNA probe to generate apurinic/apyrimidinic (AP) sites, leading to the assembly of MB-hairpin/AuNP probes on the electrode based on host-guest reaction between β-CD and MB. Meanwhile, L-cysteine (RSH) is oxidized by O to disulfide L-cystine (RSSR) and HO. In the presence of HO, Fe-N-C catalyzes the oxidation of MB to generate an amplified electrochemical signal. Notably, the Fe-N-C-catalyzed oxidation of MB is mediated by the oxidation of RSH by O instead of external HO, greatly simplifying the experimental procedures and improving the electrochemical signal. Due to the introduction of host-guest recognition, this electrochemical biosensor displays a low-background signal and high signal-to-noise ratio, enabling the one-step sensitive measurement of UDG with a detection limit of 7.4 × 10 U mL. Moreover, this biosensor can measure UDG in crude cell extracts and screen the inhibitors, providing a new platform for biomedical research.
我们开发了一种基于主体客体相互作用和铁嵌入富氮碳纳米管（Fe-N-C）的低背景电化学生物传感器，用于一步检测尿嘧啶 DNA 糖基化酶（UDG），模拟酶介导的电催化以实现信号放大。在这项工作中，首先将 Fe-N-C 固定在玻碳电极上，然后固定β-环糊精（β-CD）。我们通过将亚甲基蓝（MB）标记的发夹 DNA 组装到金纳米粒子（AuNPs）的表面来构建信号探针，从而形成 MB-发夹/AuNP 探针。由于 AuNPs 的空间位阻和发夹 DNA 的茎环结构，MB 被阻止进入电极上β-CD 的空腔。相比之下，UDG 能够从发夹 DNA 探针的茎中去除尿嘧啶，从而产生无碱基/无嘧啶（AP）位点，导致基于β-CD 与 MB 之间的主体客体反应，MB-发夹/AuNP 探针组装在电极上。同时，L-半胱氨酸（RSH）被 O 氧化为二硫化物 L-胱氨酸（RSSR）和 HO。在 HO 的存在下，Fe-N-C 催化 MB 的氧化，产生放大的电化学信号。值得注意的是，Fe-N-C 催化的 MB 氧化是由 O 氧化 RSH 介导的，而不是外部 HO，这大大简化了实验步骤并提高了电化学信号。由于引入了主体客体识别，这种电化学生物传感器显示出低背景信号和高信噪比，能够一步灵敏地测量 UDG，检测限低至 7.4×10 U mL。此外，该生物传感器可用于测量粗细胞提取物中的 UDG 并筛选抑制剂，为生物医学研究提供了一个新平台。
