Institute of Drug Research, Fujian Academy of Chinese Medicine, Fuzhou 350003, China.
Sensors (Basel). 2013 Jan 16;13(1):1064-75. doi: 10.3390/s130101064.
With an internal transcribed spacer of 18 S, 5.8 S and 26 S nuclear ribosomal DNA (nrDNA ITS) as DNA marker, we report a colorimetric approach for authentication of Pseudostellaria heterophylla (PH) and its counterfeit species based on the differentiation of the nrDNA ITS sequence. The assay possesses an unlabelled G-quadruplex DNAzyme molecular beacon (MB) probe, employing complementary sequence as biorecognition element and 1:1:1:1 split G-quadruplex halves as reporter. In the absence of target DNA (T-DNA), the probe can shape intermolecular G-quadruplex structures capable of binding hemin to form G-quadruplex-hemin DNAzyme and catalyze the oxidation of ABTS2- to blue-green ABTS•- by H(2)O(2). In the presence of T-DNA, T-DNA can hybridize with the complementary sequence to form a duplex structure, hindering the formation of the G-quadruplex structure and resulting in the loss of the catalytic activity. Consequently, a UV-Vis absorption signal decrease is observed in the ABTS2--H(2)O(2) system. The "turn-off" assay allows the detection of T-DNA from 1.0 × 10-9 to 3.0 × 10-7 mol·L-1 (R2 = 0.9906), with a low detection limit of 3.1 × 10-10 mol·L-1. The present study provides a sensitive and selective method and may serve as a foundation of utilizing the DNAzyme MB sensor for identifying traditional Chinese medicines.
以 18S、5.8S 和 26S 核核糖体 DNA（nrDNA ITS）的内部转录间隔区作为 DNA 标记，我们报告了一种比色法，用于基于 nrDNA ITS 序列的差异来鉴定假繁缕（PH）及其假冒种。该测定法具有未标记的 G-四链体 DNA 酶分子信标（MB）探针，采用互补序列作为生物识别元件和 1:1:1:1 分裂的 G-四链体半作为报告子。在没有靶 DNA（T-DNA）的情况下，探针可以形成能够结合血红素形成 G-四链体-血红素 DNA 酶并通过 H（2）O（2）催化 ABTS2-氧化为蓝绿色 ABTS•-的分子内 G-四链体结构。在存在 T-DNA 的情况下，T-DNA 可以与互补序列杂交形成双链结构，阻碍 G-四链体结构的形成，导致催化活性丧失。因此，在 ABTS2--H（2）O（2）系统中观察到 UV-Vis 吸收信号降低。“关闭”测定法允许从 1.0×10-9 到 3.0×10-7 mol·L-1（R2 = 0.9906）检测 T-DNA，检测限低至 3.1×10-10 mol·L-1。本研究提供了一种灵敏且选择性的方法，并可作为利用 DNA 酶 MB 传感器鉴定中药的基础。
