Guangxi Key Laboratory of Electrochemical and Magnetochemical Function Materials, College of Chemistry and Bioengineering, Guilin University of Technology, Guilin, 541004, Guangxi, China.
Hunan Institute of Agriculture Product Processing, Hunan Academy of Agricultural Sciences, Changsha, 410125, China.
Mikrochim Acta. 2021 Mar 15;188(4):125. doi: 10.1007/s00604-021-04777-2.
An electrochemiluminescence (ECL) DNA biosensor based on ExoIII exonuclease assistance and hybridization chain reaction (HCR) amplification technology has been constructed. ExoIII exonuclease and triple-helix DNA molecular switch are used in detecting a target in circulation. By combining HCR with AuNPs@DNA, a novel signal probe is built, which enables multiple signal amplification and the high-sensitive detection of transgenic rice BT63 DNA. The FeO@Au solution is added to a magneto-controlled glassy carbon electrode, and sulfhydryl-modified capture DNA (CP) is immobilized on FeO@Au through the Au-S bond. Mercaptoethanol is added to close sites and prevent the nonspecific adsorption of CP on the magnetron glassy carbon electrode. A target DNA is added to a constructed triple-helix DNA molecular centrifuge tube for reaction. Owing to base complementation and the reversible switching of the triple-helix DNA molecular state, the target DNA turns on the triple-helix DNA molecular switch and hybridizes with a long-strand recognition probe (RP) to form a double-stranded DNA (dsDNA). Exonuclease ExoIII is added to specifically recognize and cut the dsDNA and to release the target DNA. The target DNA strand then circulates back completely to open the multiple triple-helix DNA molecular switch, releasing a large number of signal transduction probes (STP). To hybridize with CP, a large amount of STP is added to the electrode. Finally, a AuNPs@DNA signal probe is added to hybridize with STP. H1 and H2 probes are added for the hybridization chain reaction and the indefinite extension of the primer strand on the probe. Then, tris-(bipyridyl)ruthenium(II) is added for ECL signal detection with PBS-tri-n-propylamine as the base solution. In the concentration range 1.0 × 10 to 1.0 × 10 mol/L of the target DNA, good linear relationship was achieved with the corresponding ECL signal. The detection limit is 3.6 × 10 mol/L. The spiked recovery of the rice samples range from 97.2 to 101.5%. The sensor is highly sensitive and has good selectivity, stability, and reproducibility. A novel electrochemiluminescence biosensor with extremely higher sensitivity was prepared for the determination of ultra-trace amount transgenic rice BT63 DNA. The sensitivity was significantly improved by multiple signal enhancements. Firstly, a large number of signal transduction probes are released when the triple-helix DNA molecular switch unlock after recycles assisted by ExoIII exonuclease under target BT63 DNA; and then the signal transduction probes hybridize with the signal probes of AuNPs@(DNA-HCR) produced through hybridization chain reaction. Finally, the signal probes which were embedded with a large amount of electrochemiluminescence reagent produce high luminescence intensity. The detection limit was 3.6 × 10 mol/L, which is almost the most sensitive methods reported.
基于 ExoIII 外切酶辅助和杂交链式反应 (HCR) 扩增技术构建了一种电化学发光 (ECL) DNA 生物传感器。 ExoIII 外切酶和三螺旋 DNA 分子开关用于检测循环中的靶标。通过将 HCR 与 AuNPs@DNA 结合，构建了一种新型信号探针，可实现多重信号放大和对转基因水稻 BT63 DNA 的高灵敏检测。将 FeO@Au 溶液添加到磁控玻碳电极上，并通过 Au-S 键将巯基修饰的捕获 DNA (CP) 固定在 FeO@Au 上。添加巯基乙醇以封闭位点并防止 CP 在磁控玻碳电极上的非特异性吸附。将靶 DNA 添加到构建的三螺旋 DNA 分子离心管中进行反应。由于碱基互补和三螺旋 DNA 分子状态的可逆切换，靶 DNA 打开三螺旋 DNA 分子开关并与长链识别探针 (RP) 杂交形成双链 DNA (dsDNA)。加入 ExoIII 外切酶特异性识别并切割 dsDNA，释放靶 DNA。靶 DNA 链然后完全循环以打开多个三螺旋 DNA 分子开关，释放大量信号转导探针 (STP)。为了与 CP 杂交，向电极中添加大量 STP。最后，添加 AuNPs@DNA 信号探针与 STP 杂交。添加 H1 和 H2 探针进行杂交链式反应和探针上引物链的无限延伸。然后，添加三(联吡啶)钌(II)作为电化学发光信号检测的基底溶液为 PBS-三正丙胺。在靶 DNA 的浓度范围为 1.0×10 至 1.0×10 mol/L 时，获得了与相应的 ECL 信号的良好线性关系。检测限为 3.6×10 mol/L。从水稻样品中的回收率在 97.2%至 101.5%之间。该传感器具有高灵敏度和良好的选择性、稳定性和重现性。为了测定痕量的转基因水稻 BT63 DNA，制备了一种具有极高灵敏度的新型电化学发光生物传感器。通过多重信号增强，灵敏度得到了显著提高。首先，在靶 BT63 DNA 存在下，外切酶 ExoIII 辅助循环后，三螺旋 DNA 分子开关解锁，释放大量信号转导探针；然后，信号转导探针与通过杂交链式反应产生的 AuNPs@(DNA-HCR)信号探针杂交。最后，嵌入大量电化学发光试剂的信号探针产生高发光强度。检测限为 3.6×10 mol/L，这几乎是报道的最灵敏的方法。
Biosensors (Basel). 2022-9-11
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