In this report, a facile and label-free electrochemical RNA biosensor is developed by exploiting methylene blue (MB) as an electroactive positive ligand of G-quadruplex. The electrochemical response mechanism of the nucleic acid assay was based on the change in differential pulse voltammetry (DPV) signal of adsorbed MB on the immobilized human telomeric G-quadruplex DNA with a loop that is complementary to the target RNA. Hybridization between synthetic positive control RNA and G-quadruplex DNA probe on the transducer platform rendered a conformational change of G-quadruplex to double-stranded DNA (dsDNA), and increased the redox current of cationic MB π planar ligand at the sensing interface, thereby the electrochemical signal of the MB-adsorbed duplex is proportional to the concentration of target RNA, with SARS-CoV-2 (COVID-19) RNA as the model. Under optimal conditions, the target RNA can be detected in a linear range from 1 zM to 1 μM with a limit of detection (LOD) obtained at 0.59 zM for synthetic target RNA and as low as 1.4 copy number for positive control plasmid. This genosensor exhibited high selectivity towards SARS-CoV-2 RNA over other RNA nucleotides, such as SARS-CoV and MERS-CoV. The electrochemical RNA biosensor showed DPV signal, which was proportional to the 2019-nCoV_N_positive control plasmid from 2 to 200000 copies ( = 0.978). A good correlation between the genosensor and qRT-PCR gold standard was attained for the detection of SARS-CoV-2 RNA in terms of viral copy number in clinical samples from upper respiratory specimens.

中文翻译：

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基于 RNA 响应人类端粒 G-四链体 DNA 的电化学基因传感器：SARS-CoV-2 RNA 的概念验证

在本报告中，通过利用亚甲蓝（MB）作为 G-四链体的电活性正配体，开发了一种简便且无标记的电化学 RNA 生物传感器。核酸测定的电化学响应机制是基于固定化人端粒 G-四链体 DNA 上吸附的 MB 的微分脉冲伏安 (DPV) 信号的变化，该 DNA 具有与靶 RNA 互补的环。合成的阳性对照RNA与G-四链体DNA探针在传感器平台上杂交，使G-四链体构象变化为双链DNA（dsDNA），并增加了传感界面处阳离子MB π平面配体的氧化还原电流，从而MB吸附双链体的电化学信号与目标RNA的浓度成正比，以SARS-CoV-2（COVID-19）RNA为模型。在最佳条件下，目标 RNA 的检测线性范围为 1 zM 至 1 μM，合成目标 RNA 的检测限 (LOD) 为 0.59 zM，阳性对照质粒的检测限低至 1.4 个拷贝数。该基因传感器对 SARS-CoV-2 RNA 表现出比其他 RNA 核苷酸（例如 SARS-CoV 和 MERS-CoV）更高的选择性。电化学 RNA 生物传感器显示 DPV 信号，该信号与 2019-nCoV_N_阳性对照质粒从 2 到 200000 个拷贝成正比 (= 0.978)。就上呼吸道标本临床样本中的病毒拷贝数而言，基因传感器和 qRT-PCR 金标准之间的 SARS-CoV-2 RNA 检测具有良好的相关性。