Nanoswitch-linked immunosorbent assay (NLISA) for fast, sensitive, and specific protein detection
Nanoswitch-linked immunosorbent assay (NLISA) for fast, sensitive, and specific protein detection
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DOI：
10.1073/pnas.1708148114
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发表时间：
2017-09-26
影响因子：
11.1
通讯作者：
Wong, Wesley P.
中科院分区：
文献类型：
--
作者：
Hansen, Clinton H.;Yang, Darren;Wong, Wesley P.
蛋白质检测和定量在基础研究和临床实践中都发挥着至关重要的作用。当前的检测平台范围从广泛使用的 ELISA 到更复杂、更昂贵的方法（例如数字 ELISA）。尽管取得了进步，但仍然需要一种方法，将 ELISA 的简单性和成本效益与现代方法的灵敏度和速度结合起来，以适合实验室和快速护理点应用的形式。基于 DNA 结构纳米技术的最新发展，我们推出了纳米开关连接免疫吸附测定 (NLISA)，这是一种基于易于构建的 DNA 纳米器件的检测平台，该器件在与目标蛋白结合后会改变构象，并通过凝胶电泳读出结果。 NLISA 是无表面的，包括用于纯化的动力学校对步骤，可提高灵敏度并降低交叉反应性。我们证明了对生物体液中前列腺特异性抗原的飞摩尔水平检测，以及不同登革热血清型之间以及单突变和野生型蛋白质之间的交叉反应性的降低。 NLISA 比 ELISA 更便宜，使用的样品量更少，速度更快，并且无需清洗，所需的操作步骤更少，同时还具有出色的灵敏度。我们的方法还有可能实现快速现场检测，正如我们通过使用 iPad/iPhone 摄像头进行 NLISA 成像所证明的那样。
Protein detection and quantification play critical roles in both basic research and clinical practice. Current detection platforms range from the widely used ELISA to more sophisticated, and more expensive, approaches such as digital ELISA. Despite advances, there remains a need for a method that combines the simplicity and cost-effectiveness of ELISA with the sensitivity and speed of modern approaches in a format suitable for both laboratory and rapid, point-of-care applications. Building on recent developments in DNA structural nanotechnology, we introduce the nanoswitch-linked immunosorbent assay (NLISA), a detection platform based on easily constructed DNA nanodevices that change conformation upon binding to a target protein with the results read out by gel electrophoresis. NLISA is surface-free and includes a kinetic-proofreading step for purification, enabling both enhanced sensitivity and reduced cross-reactivity. We demonstrate femtomolar-level detection of prostate-specific antigen in biological fluids, as well as reduced cross-reactivity between different serotypes of dengue and also between a single-mutation and wildtype protein. NLISA is less expensive, uses less sample volume, is more rapid, and, with no washes, includes fewer hands-on steps than ELISA, while also achieving superior sensitivity. Our approach also has the potential to enable rapid point-of-care assays, as we demonstrate by performing NLISA with an iPad/iPhone camera for imaging.