Department of Bio-Industrial Mechatronics Engineering, National Chung Hsing University, No. 145, Xingda Rd., South Dist., Taichung City 402, Taiwan.
Innovation and Development Center of Sustainable Agriculture, National Chung Hsing University, No. 145, Xingda Rd., South Dist., Taichung City 402, Taiwan.
Sensors (Basel). 2020 Jul 3;20(13):3728. doi: 10.3390/s20133728.
Developing rapid and sensitive diagnostic methods for dengue virus (DENV) infection is of prime priority because DENV infection is the most prevalent mosquito-borne viral disease. This work proposes an electrochemical impedance spectroscopy (EIS)-based genosensor for the label-free and nucleic acid amplification-free detection of extracted DENV RNA intended for a sensitive diagnosis of DENV infection. A concentration ratio of 0.04 mM 6-mercaptohexanoic acid (MHA) to 1 mM 6-mercapto-1-hexanol (MCH) was selected to modify thin-film gold electrodes as a link to control the coverage of self-designed probe DNA (pDNA) at a density of 4.5 ± 0.4 × 10 pDNA/cm. The pDNA/MHA/MCH-modified genosensors are proven to improve the hybridization efficiency of a synthetic 160-mer target DNA (160mtDNA) with a 140-mer electrode side overhang as compared to other MHA/MCH ratio-modified genosensors. The MHA(0.04 mM)/MCH(1 mM)-modified genosensors also present good hybridization efficiency with the extracted DENV serotype 1 (DENV1) RNA samples, having the same electrode side overhangs with the 160mtDNA, showing a low detection limit of 20 plaque forming units (PFU)/mL, a linear range of 10-10 PFU/mL and good selectivity for DENV1. The pDNA density-controlled method has great promise to construct sensitive genosensors based on the hybridization of extracted DENV nucleic acids.
开发用于登革热病毒（DENV）感染的快速和敏感诊断方法至关重要，因为 DENV 感染是最普遍的蚊媒病毒病。本工作提出了一种基于电化学阻抗谱（EIS）的基因传感器，用于无标记和无核酸扩增检测提取的 DENV RNA，旨在对 DENV 感染进行灵敏诊断。选择浓度比为 0.04 mM 巯基己酸（MHA）和 1 mM 巯基-1-己醇（MCH）来修饰薄膜金电极，作为控制自设计探针 DNA（pDNA）覆盖率的连接体，其密度为 4.5±0.4×10 pDNA/cm。事实证明，pDNA/MHA/MCH 修饰的基因传感器可以提高合成的 160 个碱基对目标 DNA（160mtDNA）与具有 140 个碱基对电极悬垂的杂交效率，与其他 MHA/MCH 比修饰的基因传感器相比。MHA（0.04 mM）/MCH（1 mM）修饰的基因传感器也与具有相同电极悬垂的提取的 DENV 血清型 1（DENV1）RNA 样本表现出良好的杂交效率，显示出 20 噬菌斑形成单位（PFU）/mL 的低检测限、10-10 PFU/mL 的线性范围和对 DENV1 的良好选择性。pDNA 密度控制方法有望基于提取的 DENV 核酸杂交构建灵敏的基因传感器。
