Department of Food Quality and Safety, College of Food Science and Engineering, Jilin University, Changchun, 130062, China.
Chongqing Research Institute, Jilin University, Changchun, People's Republic of China.
Anal Bioanal Chem. 2022 Sep;414(22):6581-6590. doi: 10.1007/s00216-022-04218-w. Epub 2022 Jul 13.
Based on a Pb-specific 8-17 DNAzyme-induced catalytic hairpin assembly (CHA), a simple signal-on fluorescence strategy for lead ion detection was established. 8-17 DNAzyme was used as the recognition element of Pb, which catalyzed the cleavage of the RNA base embedded in the DNA substrate strand, while releasing part of the substrate strand (S') as CHA initiator. And two hairpin probes (H1 and H2-FQ) were designed according to the sequence of S' for CHA, in which H2-FQ was labeled with the fluorophore FAM and quencher BHQ-1 as fluorescent "molecular switch" based on fluorescence resonance energy transfer (FRET). In the presence of Pb, the CHA reaction was triggered to form a large number of H1-H2 complexes, enabling enzyme-free isothermal amplification and a signal-on fluorescence strategy. In the concentration range of 0.5-1000 nM, the fluorescence signal increases with the increase of Pb concentration. The quantitative detection limit of Pb by this method is 0.5 nM, which has better detection performance compared with the FQ-labeled 8-17 DNAzyme method. The established biosensor exhibits good specificity and can be effectively used for the detection of Pb in real samples of river water and grass carp. Through ingenious nucleic acid sequence design, DNAzyme and CHA reactions are integrated to realize the enzyme-free isothermal amplifications and sensitive detection of Pb, which holds potential versatility in food supervision and environmental monitoring.
基于 Pb 特异性 8-17 DNA 酶诱导的催化发夹组装(CHA),建立了一种用于检测铅离子的简单信号开启荧光策略。8-17 DNA 酶被用作 Pb 的识别元件,它可以催化嵌入 DNA 底物链中的 RNA 碱基的切割,同时释放部分底物链(S')作为 CHA 的起始子。根据 S'的序列设计了两个发夹探针(H1 和 H2-FQ),用于 CHA,其中 H2-FQ 被荧光团 FAM 和淬灭剂 BHQ-1 标记,作为基于荧光共振能量转移(FRET)的荧光“分子开关”。在存在 Pb 的情况下,CHA 反应被触发以形成大量的 H1-H2 复合物,从而实现无酶等温扩增和信号开启荧光策略。在 0.5-1000 nM 的浓度范围内,荧光信号随 Pb 浓度的增加而增加。该方法对 Pb 的定量检测限为 0.5 nM,与 FQ 标记的 8-17 DNA 酶方法相比,具有更好的检测性能。所建立的生物传感器具有良好的特异性,可有效用于河水和草鱼实际样本中 Pb 的检测。通过巧妙的核酸序列设计,将 DNA 酶和 CHA 反应集成在一起,实现了无酶等温扩增和对 Pb 的灵敏检测,在食品监管和环境监测方面具有潜在的多功能性。