Key Laboratory of Chemistry of Northwestern Plant Resources and Key Laboratory for Natural Medicine of Gansu Province, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Lanzhou 730000, China.
Key Laboratory of Chemistry of Northwestern Plant Resources and Key Laboratory for Natural Medicine of Gansu Province, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Lanzhou 730000, China.
Biosens Bioelectron. 2015 Jun 15;68:550-555. doi: 10.1016/j.bios.2015.01.054. Epub 2015 Jan 23.
DNA plays important regulatory roles in many life activities. Here, we have developed a novel label-free, ultrasensitive and specific chemiluminescence (CL) assay protocol for DNA detection based on hairpin assembly-triggered cyclic activation of a DNA machine. The system involves two hairpin structures, H1 and H2. Firstly, a target DNA binds with and opens the hairpin structure of H1. Then, H2 hybridizes with H1 and displaces the target DNA, which is used to trigger another new hybridization cycle between H1 and H2, leading to the generation of numerous H1-H2 complexes. The generated H1-H2 complexes are further activated with the help of polymerase and nicking enzyme, continuously yielding a large amount of G-riched DNA fragments. The G-riched DNA fragment products interact with hemin to form the activated HRP-mimicking DNAzymes that can catalyze the oxidation of luminol by H2O2 to produce strong CL signal resulting in an amplified sensing process. Our newly proposed homogeneous assay enables the quantitative measurement of p53 DNA (as a model) with a detection limit of 0.85 fM, which is at least 5 orders of magnitude lower than that of traditional unamplified homogeneous optical approaches. Moreover, this assay exhibits high discrimination ability even against a single base mismatch. In addition, this strategy is also capable of detecting p53 DNA in complex biological samples. The proposed sensing approach might hold a great promise for further applications in biomedical research and early clinical diagnosis.
DNA 在许多生命活动中发挥着重要的调节作用。在这里，我们开发了一种基于发夹组装触发 DNA 机器循环激活的新型无标记、超灵敏和特异的化学发光（CL）检测方法用于 DNA 检测。该系统涉及两个发夹结构，H1 和 H2。首先，靶 DNA 与 H1 的发夹结构结合并打开它。然后，H2 与 H1 杂交并置换靶 DNA，这被用来触发 H1 和 H2 之间的另一个新的杂交循环，导致大量 H1-H2 复合物的产生。在聚合酶和缺口酶的帮助下，生成的 H1-H2 复合物被进一步激活，不断产生大量富含 G 的 DNA 片段。富含 G 的 DNA 片段产物与血红素相互作用形成被激活的 HRP 模拟 DNA 酶，它可以催化 H2O2 氧化鲁米诺产生强 CL 信号，从而实现放大的传感过程。我们新提出的均相测定法能够定量测量 p53 DNA（作为模型），检测限为 0.85 fM，比传统的非放大均相光学方法至少低 5 个数量级。此外，该测定法即使针对单个碱基错配也具有高分辨能力。此外，这种策略还能够检测复杂生物样品中的 p53 DNA。该传感方法在生物医学研究和早期临床诊断中的进一步应用可能具有很大的前景。
