School of Materials Science and Engineering, Nanyang Technological University, 50 Nanyang Avenue, 639798, Singapore; Department of Biotechnology Engineering, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel; NTU-HUJ-BGU CREATE Programme, 1 Create Way, Research Wing #02-06 to 08, 138602, Singapore.
School of Materials Science and Engineering, Nanyang Technological University, 50 Nanyang Avenue, 639798, Singapore; Department of Biotechnology Engineering, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel; NTU-HUJ-BGU CREATE Programme, 1 Create Way, Research Wing #02-06 to 08, 138602, Singapore; The National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel; The Ilse Katz Center for Meso and Nanoscale Science and Technology, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel.
Biosens Bioelectron. 2017 Jan 15;87:572-578. doi: 10.1016/j.bios.2016.08.044. Epub 2016 Aug 18.
A new colorimetric immunoassay concept, utilizing conventional lateral flow membranes (e.g., conjugation, sample, absorption and nitrocellulose), were placed in a different configuration in a stacking manner, where the liquid sample that may contain the analyte diffuses from the bottom to the upper-most layer. The key element of this proprietary technology is a capture layer, where a nitrocellulose membrane is modified with the target analyte of interest, namely in this study target Escherichia coli. During the immunoassay operation, samples contaminated with the target bacteria will conjugate to their corresponding HRP-antibodies laying in wait and the immune-target measurand complex flows by capillarity towards the upper-most layer to generate a colorimetric signal (positive answer) through an enzymatic reaction. In target-free samples, previously immobilized target bacteria on the capture layer will prevent the HRP-labeled anti-target antibodies from migrating to the upper-most layer, where the enzymatic substrate lays in wait. After optimization, the sensitivity of this approach was found to be 1,000 folds higher than ELISAs (10cellsmL). The advantages of the stacked pad assay include: miniaturization, operational simplicity, fast response time (less than 5min), useful sensitivity.
一种新的比色免疫分析概念，利用传统的横向流动膜（例如，结合、样品、吸收和硝酸纤维素）以堆叠的方式放置在不同的配置中，其中可能含有分析物的液体样品从底部扩散到最上层。这项专有技术的关键要素是一个捕获层，其中硝酸纤维素膜用目标分析物（即本研究中的目标大肠杆菌）进行修饰。在免疫分析操作过程中，受污染的样品与潜伏的目标细菌的相应 HRP 抗体结合，免疫目标测定物复合物通过毛细作用流向最上层，通过酶反应产生比色信号（阳性结果）。在无目标的样品中，先前固定在捕获层上的目标细菌会阻止 HRP 标记的抗目标抗体迁移到最上层，那里潜伏着酶底物。经过优化，这种方法的灵敏度比 ELISA 高 1000 倍（10 个细胞/ml）。堆叠垫检测法的优点包括：小型化、操作简单、快速响应时间（少于 5 分钟）、灵敏度高。
