Makela Megan, Lin Zhihai, Lin Pao Tai
Department of Materials Science and Engineering, Texas A&M University, College Station, Texas 77843.
Department of Electrical and Computer Engineering, Texas A&M University, College Station, Texas 77843.
Giant (Oxf). 2023 Jun 19:100173. doi: 10.1016/j.giant.2023.100173.
Rapid detection of unlabeled SARS-CoV-2 genetic target was demonstrated using a competitive displacement hybridization assay made by a nanostructured anodized alumina oxide (AAO) membrane. The assay applied the toehold-mediated strand displacement reaction. The nanoporous surface of the membrane was functionalized with a complementary pair consisting of Cy3-labeled probe and quencher-labeled nucleic acids through a chemical immobilization process. In the presence of the unlabeled SARS-CoV-2 target, the quencher-tagged strand of the immobilized probe-quencher duplex was separated from the Cy3-modifed strand. A stable probe-target duplex formed and regained a strong fluorescence signal, thus enabling real-time and label-free SARS-CoV-2 detection. Assay designs with different numbers of base pair (bp) matches were synthesized to compare their affinities. Because of the large surface of a free-standing nanoporous membrane, two orders enhancement of the fluorescence was observed, where the detection limit of the unlabeled concentration can be improved to 1 nM. The assay was miniaturized by integrating a nanoporous AAO layer onto an optical waveguide device. The detection mechanism and the sensitivity improvement of the AAO-waveguide device were illustrated from the finite difference method (FDM) simulation and the experimental results. Light-analyte interaction was further improved due to the presence of the AAO layer, which created an intermediate refractive index and enhanced the waveguide's evanescent field. Our competitive hybridization sensor is an accurate and label-free testing platform applicable to the deployment of compact and sensitive virus detection strategies.
利用纳米结构化阳极氧化铝（AAO）膜制备的竞争性置换杂交分析法，实现了对未标记的严重急性呼吸综合征冠状病毒2（SARS-CoV-2）基因靶点的快速检测。该分析方法应用了引发链介导的链置换反应。通过化学固定过程，用由Cy3标记的探针和淬灭剂标记的核酸组成的互补对将膜的纳米多孔表面功能化。在未标记的SARS-CoV-2靶点存在的情况下，固定的探针-淬灭剂双链体中带有淬灭剂标签的链与Cy3修饰的链分离。形成了稳定的探针-靶点双链体并恢复了强荧光信号，从而实现了实时且无标记的SARS-CoV-2检测。合成了具有不同碱基对（bp）匹配数的分析设计，以比较它们的亲和力。由于独立纳米多孔膜的大表面积，观察到荧光增强了两个数量级，其中未标记浓度的检测限可提高到1 nM。通过将纳米多孔AAO层集成到光波导装置上，该分析方法得以小型化。从有限差分法（FDM）模拟和实验结果说明了AAO-波导装置的检测机制和灵敏度提高情况。由于AAO层的存在，光与分析物的相互作用进一步得到改善，AAO层产生了中间折射率并增强了波导的倏逝场。我们的竞争性杂交传感器是一个准确且无标记的测试平台，适用于部署紧凑且灵敏的病毒检测策略。
