Key Laboratory of Luminescence and Real-Time Analysis, Ministry of Education, School of Chemistry and Chemical Engineering, Southwest University, Chongqing, 400715, People's Republic of China.
Mikrochim Acta. 2019 Jul 29;186(8):582. doi: 10.1007/s00604-019-3666-4.
A DNA dendrimer was constructed by combination of rolling circle amplification (RCA) and hybridization chain reaction (HCR). A fluorescence resonance energy transfer (FRET) pair consisting of 6-carboxyfluorescein (FAM) and 5-carboxytetramethylrhodamine (TAMRA) was then created as a signaling probe for the ultrasensitive detection of Pb. Firstly, the DNAzyme released by Pb-induced recycling amplification as a primer induces RCA to open two different hairpins for generating repeated "Y" structures. Next, two other hairpins (labeled with FAM and TAMRA, respectively) are opened by the "Y" structures to trigger the HCR. As a result, a DNA dendrimer is generated. It is high loading with FRET pair and also promotes FRET pair mutually close to produce a remarkable FRET signal. Hence, ultrasensitive detection of Pb is accomplished by measurement of the ratio of the yellow fluorescence of TAMRA (peaking at 588 nm) and the green fluorescence of FAM (at 525 nm). The method works in the 0.001 to 10 nM Pb concentration range and has a 0.3 pM detection limit. It was applied to determination of intracellular Pb with convincing performance. Graphical abstractSchematic representation of novel DNA dendrimer produced by the combination of rolling circle amplification (RCA) and hybridization chain reaction (HCR) for immobilization of the fluorescence resonance energy transfer (FRET) pair as signal probe for sensitive determination of Pb.
一种 DNA 树状聚合物通过滚环扩增（RCA）和杂交链式反应（HCR）的结合构建而成。然后创建了一个由 6-羧基荧光素（FAM）和 5-羧基四甲基罗丹明（TAMRA）组成的荧光共振能量转移（FRET）对，作为超灵敏检测 Pb 的信号探针。首先，Pb 诱导的循环放大释放的 DNA 酶作为引物诱导 RCA 打开两个不同的发夹，以产生重复的“Y”结构。接下来，两个其他的发夹（分别标记有 FAM 和 TAMRA）被“Y”结构打开，触发 HCR。结果，生成了 DNA 树状聚合物。它高度负载 FRET 对，并促进 FRET 对相互靠近，产生显著的 FRET 信号。因此，通过测量 TAMRA 的黄色荧光（在 588nm 处峰值）和 FAM 的绿色荧光（在 525nm 处）的比值来实现对 Pb 的超灵敏检测。该方法在 0.001 至 10nM Pb 浓度范围内有效，检测限为 0.3pM。它已成功应用于细胞内 Pb 的测定，具有令人信服的性能。
