Abstract：
G-quadruplex/thioflavin T (G4/THT) is one of the ideal label-free fluorescent light-emitting elements in the field of biosensors due to its good programmability and adaptability. However, the unsatisfactory luminous efficiency of single-molecule G4/THT limits its more practical applications. Here, we developed a G4 embedded semi-catalytic hairpin assembly (G4-SCHA) reaction by rationally modifying the traditional CHA reaction, and combined with the invasive reaction, supplemented by magnetic separation technology, for label-free sensitive detection of single nucleotide polymorphisms (SNPs). The invasive reaction enabled specific recognition of single-base mutations in DNA sequences as well as preliminary signal cycle amplification. Then, magnetic separation was used to shield the false positive signals. Finally, the G4-SCHA was created for secondary amplification and label-free output of the signal. This dual-signal amplified label-free biosensor has been shown to detect mutant targets as low as 78.54 fM. What\'s more, this biosensor could distinguish 0.01 % of the mutant targets from a mixed sample containing a large number of wild-type targets. In addition, the detection of real and complex biological samples also verified the practical application value of this biosensor in the field of molecular design breeding. Therefore, this study improves a label-free fluorescent light-emitting element, and then proposes a simple, efficient and universal label-free SNP biosensing strategy, which also provides an important reference for the development of other G4/THT based biosensors.
摘要：
G-四链体/硫黄素T(G4/THT)具有良好的可编程性和适应性,是生物传感器领域理想的无标记荧光发光元件之一。然而,单分子G4/THT不理想的发光效率限制了其更实际的应用。这里,我们通过对传统的CHA反应进行合理的修饰，开发了G4嵌入式半催化发夹组装（G4-SCHA）反应，结合侵入性反应，辅以磁选技术，用于单核苷酸多态性（SNP）的无标记灵敏检测。侵入性反应能够特异性识别DNA序列中的单碱基突变以及初步的信号循环扩增。然后，磁分离用于屏蔽假阳性信号。最后,G4-SCHA用于二次扩增和无标记输出信号.这种双信号放大的无标记生物传感器已显示可检测低至78.54fM的突变靶标。更重要的是，这种生物传感器可以从含有大量野生型靶标的混合样品中区分出0.01％的突变靶标。此外,真实和复杂生物样品的检测也验证了该生物传感器在分子设计育种领域的实际应用价值。因此,这项研究改进了无标签荧光发光元件，然后提出了一个简单的，高效和通用的无标记SNP生物传感策略，也为其他G4/THT基生物传感器的研制提供了重要参考。
