Beijing Key Laboratory for Bioengineering and Sensing Technology; School of Chemistry and Biological Engineering, University of Science and Technology Beijing, 30 Xueyuan Road, Haidian District, Beijing 100083, China.
Division of Analytical and Environmental Toxicology, Department of Laboratory Medicine and Pathology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta T6G 2G3, Canada.
Anal Chem. 2024 Jul 23;96(29):12022-12029. doi: 10.1021/acs.analchem.4c02087. Epub 2024 Jul 13.
Disease diagnostics and surveillance increasingly highlight the importance of portable, cost-effective, and sensitive point-of-care (POC) detection of nucleic acids. Here, we report a CRISPR/Cas13a-responsive and RNA-bridged DNA hydrogel capillary sensor for the direct and visual detection of specific RNA with high sensitivity. The capillary sensor was simply prepared by loading RNA-cross-linking DNA hydrogel film (∼0.2 mm ± 0.02 mm) at the end of a capillary. When CRISPR/Cas13a specifically recognizes the target RNA, the RNA bridge in the hydrogel film is cleaved by the -cleavage activity of CRISPR/Cas13a, increasing the permeability of the hydrogel film. Different concentrations of target RNA activate different amounts of Cas13a, cleaving different amounts of the RNA bridge in the hydrogel and causing corresponding changes in the permeability of the hydrogel. Therefore, samples containing different amounts of the target RNA travel to different distances in the capillary. Visual reading of the distance provides quantitative detection of the RNA target without the need for any nucleic acid amplification or auxiliary equipment. The technique was successfully used for the determination of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in clinical nasopharyngeal (NP) swab and saliva samples. Easily quantifiable distance using a ruler eliminates the need for any optical or electrochemical detection equipment, making this assay potentially useful for POC and on-site applications.
疾病诊断和监测越来越强调便携式、具有成本效益且灵敏的即时检测（POC）对核酸的重要性。在这里，我们报告了一种基于 CRISPR/Cas13a 反应和 RNA 桥接 DNA 水凝胶的毛细管传感器，用于直接和可视化地高灵敏度检测特定 RNA。毛细管传感器通过将 RNA 交联 DNA 水凝胶膜（∼0.2 毫米±0.02 毫米）加载到毛细管的末端来简单制备。当 CRISPR/Cas13a 特异性识别靶 RNA 时，水凝胶膜中的 RNA 桥被 CRISPR/Cas13a 的 -切割活性切割，增加水凝胶膜的通透性。不同浓度的靶 RNA 激活不同数量的 Cas13a，切割水凝胶中不同数量的 RNA 桥，并导致水凝胶通透性相应变化。因此，含有不同量靶 RNA 的样品在毛细管中移动到不同的距离。无需任何核酸扩增或辅助设备即可通过对距离的目视读数进行定量检测。该技术成功用于临床鼻咽（NP）拭子和唾液样本中严重急性呼吸综合征冠状病毒 2（SARS-CoV-2）RNA 的测定。使用尺子进行可轻松量化的距离测量，无需任何光学或电化学检测设备，使得该检测方法在 POCT 和现场应用中具有潜在的用途。
