State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Key Laboratory for Bio-Nanotechnology and Molecular Engineering of Hunan Province, Changsha 410082, China.
Anal Chem. 2013 Aug 20;85(16):7689-95. doi: 10.1021/ac4001157. Epub 2013 Aug 9.
A novel, high sensitive, and specific DNA assay based on gold nanoparticle (AuNP) colorimetric detection and hybridization chain reaction (HCR) amplification has been demonstrated in this article. Two hairpin auxiliary probes were designed with single-stranded DNA (ssDNA) sticky ends which stabilize AuNPs and effectively prevent them from salt-induced aggregation. The target DNA hybridized with the hairpin auxiliary probes and triggered the formation of extended double-stranded DNA (dsDNA) polymers through HCR. As a result, the formed dsDNA polymers provide less stabilization without ssDNA sticky ends, and AuNPs undergo aggregation when salt concentration is increased. Subsequently, a pale purple-to-blue color variation is observed in the colloid solution. The system is simple in design and convenient in operation. The novel strategy eliminates the need for enzymatic reactions, separation processes, chemical modifications, and sophisticated instrumentation. The detection and discrimination process can be seen with the naked eye. The detection limit of this method is lower than or at least comparable to previous AuNP-based methods. Importantly, the protocol offers high selectivity for the determination between perfectly matched target oligonucleotides and targets with single base-pair mismatches.
本文提出了一种基于金纳米粒子（AuNP）比色检测和杂交链式反应（HCR）扩增的新型、高灵敏、高特异性的 DNA 检测方法。设计了两条带有单链 DNA（ssDNA）粘性末端的发夹辅助探针，这些粘性末端可以稳定 AuNP，并有效防止其因盐诱导聚集。目标 DNA 与发夹辅助探针杂交，并通过 HCR 触发延伸双链 DNA（dsDNA）聚合物的形成。结果，形成的 dsDNA 聚合物由于没有 ssDNA 粘性末端，提供的稳定性降低，当盐浓度增加时，AuNP 发生聚集。随后，胶体溶液中观察到从浅紫色到蓝色的颜色变化。该系统设计简单，操作方便。该新策略消除了对酶反应、分离过程、化学修饰和复杂仪器的需求。可以用肉眼观察到检测和区分过程。该方法的检测限低于或至少与以前基于 AuNP 的方法相当。重要的是，该方案在完全匹配的靶寡核苷酸和具有单个碱基对错配的靶之间的测定中提供了高选择性。
