Abstract：
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 (R2 = 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.
摘要：
在这份报告中,通过利用亚甲基蓝（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基于固定的人端粒G-四链体DNA上吸附的MB的差分脉冲伏安法（DPV）信号的变化，该DNA具有与靶RNA互补的环。在换能器平台上，合成阳性对照RNA与G-四链体DNA探针之间的杂交使G-四链体构象变化为双链DNA（dsDNA），并增加了阳离子MBπ平面配体在传感界面处的氧化还原电流，因此，MB吸附的双链体的电化学信号与靶RNA的浓度成正比，以SARS-CoV-2(COVID-19)RNA为模型。在最优条件下,目标RNA可以在1μM至1μM的线性范围内检测，合成目标RNA的检测限（LOD）为0.59μM，阳性对照质粒的拷贝数低至1.4。该基因传感器对SARS-CoV-2RNA的选择性高于其他RNA核苷酸，如SARS-CoV和MERS-CoV。电化学RNA生物传感器显示DPV信号,与2至200000个拷贝的2019-nCoV_N_阳性对照质粒成正比(R2=0.978)。在上呼吸道标本的临床样品中，SARS-CoV-2RNA的病毒拷贝数检测中，基因传感器与qRT-PCR金标准之间具有良好的相关性。
