Southeast Asia Disaster Prevention Research Initiative (SEADPRI), Institute for Environment and Development (LESTARI), Universiti Kebangsaan Malaysia, 43600, UKM Bangi, Selangor Darul Ehsan, Malaysia.
Faculty of Engineering and Built Environment, Universiti Kebangsaan Malaysia, 43600, UKM Bangi, Selangor Darul Ehsan, Malaysia.
Talanta. 2024 Jul 1;274:125916. doi: 10.1016/j.talanta.2024.125916. Epub 2024 Mar 18.
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 (R = 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.
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。该核酸测定的电化学响应机制基于吸附在固定化人类端粒 G-四链体 DNA 上的 MB 的差分脉冲伏安法（DPV）信号变化，该 DNA 上带有与目标 RNA 互补的环。合成正对照 RNA 与传感器平台上的 G-四链体 DNA 探针之间的杂交导致 G-四链体构象发生变化，形成双链 DNA（dsDNA），并增加了阳离子 MB π 平面配体在传感界面上的氧化还原电流，从而 MB 吸附双链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 可以在 1 zM 至 1 μM 的线性范围内检测到，对于合成目标 RNA 的检测限（LOD）为 0.59 zM，对于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 表现出高选择性，对其他 RNA 核苷酸如 SARS-CoV 和 MERS-CoV 具有较高的选择性。电化学 RNA 生物传感器显示出与 qRT-PCR 金标准的良好相关性，可用于检测临床样本中来自上呼吸道标本的 2019-nCoV_N 阳性对照质粒的 SARS-CoV-2 RNA。
