Abstract：
Compared to conventional nucleic acid detection methods, label-free single nucleotide polymorphism (SNP) detection presents challenging due to the necessity of discerning single base mismatches, especially in the field of enzyme-free detection. In this study, we introduce a novel bulged-type DNA duplex probe designed to significantly amplify single-base differences. This probe is integrated with programmable DNA-based nanostructures to develop a sensitive, label-free biosensor for nonenzymatic SNP detection. The duplex probe with one bulge could selectively identify wild-typed DNA (WT) and mutant-type DNA (MT) based on a competitive strand displacement reaction mechanism. The hyperbranched HCR (HHCR) by incorporating of hairpin DNA into the DNA tetrahedron and surface-tethering on the portable screen printing electrode (SPCE) significantly favor the formation of negatively charged DNA nanostructure. We harnessed strong repulsion of DNA nanostructure towards the electroactive [Fe(CN)₆]³⁻/⁴⁻ in combination with electrochemical technique to create a label-free biosensor. This simple, enzyme-free and label-free biosensor could detect MT with a detection limit of 56 aM, even in multiple sequence backgrounds. The study served as the proof-of-concept for the integration of enzyme-free competitive mechanism and label-free strategy, which can be extended as a powerful tool to various fields.
摘要：
与常规核酸检测方法相比,无标记单核苷酸多态性（SNP）检测提出了挑战，由于辨别单碱基错配的必要性，特别是在无酶检测领域。在这项研究中,我们引入了一种新型的凸起型DNA双链体探针,旨在显着扩增单碱基差异.该探针与可编程的基于DNA的纳米结构集成在一起，以开发一种灵敏的，用于非酶SNP检测的无标记生物传感器。具有一个凸起的双链体探针可以基于竞争性链置换反应机制选择性地识别野生型DNA（WT）和突变型DNA（MT）。超支化HCR（HHCR）通过将发夹DNA掺入DNA四面体并在便携式丝网印刷电极（SPCE）上进行表面束缚，显着有利于形成带负电荷的DNA纳米结构。我们结合电化学技术，利用DNA纳米结构对电活性[Fe（CN）]的强烈排斥作用，创建了无标记的生物传感器。这个简单的，无酶无标记生物传感器可以检测MT，检测限为56aM，即使在多个序列背景中。该研究为无酶竞争机制和无标签策略的整合提供了概念验证，它可以作为一个强大的工具扩展到各个领域。
