Exonuclease III-assisted strand displacement reaction-driven cyclic generation of G-quadruplex strategy for homogeneous fluorescent detection of melamine
Exonuclease III-assisted strand displacement reaction-driven cyclic generation of G-quadruplex strategy for homogeneous fluorescent detection of melamine
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DOI：
10.1016/j.talanta.2019.05.020
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发表时间：
2019-10-01
期刊：
影响因子：
6.1
通讯作者：
Geng, Jia
中科院分区：
文献类型：
--
作者：
Chen, Piaopiao;Huang, Ke;Geng, Jia
发展灵活、灵敏、选择性强、操作简便的三聚氰胺检测技术具有重要意义。在这里，我们提出了一种均相关闭模式的荧光策略，检测三聚氰胺通过耦合的过程中的核酸外切酶III辅助（Exo III）的扩增与链置换反应（SDR）驱动的组装的DNA G-四链体结构。三聚氰胺（M）可通过氢键与胸腺嘧啶（T）碱基结合形成T-M-T结构，抑制后续核酸扩增反应和G-四链体结构的形成。DNA链（G1）既作为识别探针又作为信号探针，从而大大简化了实验设计和操作。此外，为了提高检测灵敏度，巧妙地设计了包含G-四链体序列（G2）和部分杂交的信号触发链（P1）的双链DNA片段（dsDNA，P1-G2）。在SDR和Exo III辅助扩增后，G1-DNA和G2-DNA被释放，并开始G-四链体结构的循环产生。产生的DNA G-四链体结合到NMM（一种荧光染料，N-甲基中卟啉IX），提供增强的荧光信号。这允许在低至25 fM的浓度下高度灵敏地检测三聚氰胺。所提出的三聚氰胺浓度的荧光检测方法在100 fM至100 pM范围内显示出良好的线性关系，这表明比非酶辅助传感系统（检测限为15 pM）的性能有所改善。此外，与其他物质相比，该方法对目标分子三聚氰胺表现出高选择性。将该方法用于真实的牛奶样品中三聚氰胺的测定，获得了满意的结果。本研究开发的新策略可能被认为是一种高灵敏度、简单、选择性和准确测定小分子的潜在途径。
Development of flexible, sensitive, selective and simple melamine detection is in high demand and of great significance. Here, we proposed a homogeneous turn-off mode fluorescent strategy to detect melamine by coupling the process of exonuclease III-assisted (Exo III) amplification with the strand displacement reaction (SDR)-driven assembly of DNA G-quadruplex structures. Melamine (M) could bind to the thymine (T) base through hydrogen bonding to form a T-M-T structure, which inhibits the subsequent nucleic acid amplification reaction and formation of the G-quadruplex structure. DNA strand (G1) served as both a recognition probe and a signal probe, thus greatly simplifying the experimental design and operation. Furthermore, to improve the detection sensitivity, an ingeniously designed segment of double-stranded DNA (dsDNA, P1-G2) that contained the G-quadruplex sequence (G2) and a partially hybridized signal trigger strand (P1) was added to the system. Following the SDR and Exo III-assisted amplification, G1-DNA and G2-DNA were released and the cyclic production of G-quadruplex structures was initiated. The generated DNA G-quadruplex bound to the NMM (a fluorescent dye, N-methyl mesoporphyrin IX), providing enhanced fluorescence signals. This allowed for the highly sensitive detection of melamine at concentrations as low as 25 fM. The proposed fluorescent detection method of melamine concentration displayed a good linear relationship ranging from 100 fM to 100 pM, which showed improved performance over that of the non-enzyme-assisted sensing system (limit of detection is 15 pM). Additionally, this method exhibited a high selectivity towards the target molecule, melamine, in comparison to other substances. Furthermore, the method was used for the assay of melamine in real milk samples and satisfactory experiment results were obtained. The novel strategy developed in this research may be considered as a potential route for highly sensitive, simple, selective and accurate assay of small molecules.