設施農業：智慧感測【生物產業機電工程學系/吳靖宙特聘教授】
| 論文篇名 | 英文：A Label-Free Impedimetric Genosensor for the Nucleic Acid Amplification-Free Detection of Extracted RNA of Dengue Virus 中文：免標定式基因感測器對登革熱病毒萃取RNA進行免核酸放大之檢測 |
| 期刊名稱 | SENSORS |
| 發表年份,卷數,起迄頁數 | 2020, 20 (13), 3728 |
| 作者 | Wu, Ching-Chou(吳靖宙)*; Yen, Hao-Yu; Lai, Lu-Ting; Perng, Guey-Chuen; Lee, Cheng-Rei; Wu, Shuenn-Jue |
| DOI | 10.3390/s20133728 |
| 中文摘要 | 開發針對登革熱病毒(DENV)的快速靈敏檢測法為防治DENV傳染的首要工作之一。本研究提出了一種基於電化學組抗頻譜分析(EIS)的基因感測器，用於無標定和無核酸放大程序直接對萃取後DENV RNA進行檢測。利用0.04 mM 6-巰基己酸（MHA）與1 mM 6-巰基-1-己醇（MCH）濃度混合液修飾薄膜金電極，可控制探針DNA(pDNA)的表面覆蓋率密度至4.5 ± 0.4*10-11 pDNA/cm2。相較於其他MHA/MCH的比率修飾膜，此pDNA/MHA/MCH-修飾之基因感測器已被證實能增進160-mer 目標DNA(160mtDNA)與pDNA的雜合。該基因感測器也能對萃取後的DENV血清型1(DENV1) RNA樣本進行雜合與檢測，其檢測極限為20 PFU/mL，而線性範圍落在102–105 PFU/mL，且具有良好的DENV1選別性。採用MHA/MCH混合修飾藉以控制pDNA密度的方法，對建構一個高靈敏性DENV核酸感測器有著令人期待的前景。 |
| 英文摘要 | 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*10-11 pDNA/cm2. 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 102–105 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. |
圖 (a)在pDNA/MHA(0.04 mM)/MCH(1 mM)/Au 基因感測器，在經過20分鐘的RNA片段雜合後，RNA的濃度從(ii)102, (iii)103, (iv)104, (v) 105, 到(vi)106的Nyquist plots圖 。圖 (b)此基因感測器相對應的檢量線(n=3)。
