Droplet-Free Digital Enzyme-Linked Immunosorbent Assay Based on a Tyramide Signal Amplification System
Droplet-Free Digital Enzyme-Linked Immunosorbent Assay Based on a Tyramide Signal Amplification System
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DOI：
10.1021/acs.analchem.6b01148
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发表时间：
2016-07-19
影响因子：
7.4
通讯作者：
Suzuki, Seigo
中科院分区：
文献类型：
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作者：
Akama, Kenji;Shirai, Kentaro;Suzuki, Seigo
数字酶联免疫吸附测定（ELISA）是一种单分子计数技术，是最灵敏的免疫测定方法之一。该技术的关键是将单个目标分子的酶反应产物浓缩到飞升液滴中。本研究提出了一种不需要液滴的新型数字ELISA；相反，酶反应产物浓缩使用酰胺信号放大系统。在我们的方法中，酪酰胺底物与用免疫复合物标记的辣根过氧化物酶（HRP）反应，底物转化为短寿命的自由基中间体。通过调节HRP-酪酰胺反应中的微球浓度，利用自由移动的微球进行反应，酪酰胺自由基仅沉积在HRP标记的微球上，而不会扩散到其他微球上。因此，荧光信号被定位在珠子的一部分上，使得以数字方式计算标记珠子的数量成为可能。结果表明，该方法对乙型肝炎表面抗原的检测限为0.09 mIU/mL (139 aM)，动态范围超过4个数量级。所获得的检测限比常规ELISA的灵敏度高20倍。我们的方法在检测超低浓度蛋白质生物标志物的简单体外诊断系统中具有潜在的应用前景。
Digital enzyme-linked immunosorbent assay (ELISA) is a single molecule counting technology and is one of the most sensitive immunoassay methods. The key aspect of this technology is to concentrate enzyme reaction products from a single target molecule in femtoliter droplets. This study presents a novel Digital ELISA that does not require droplets; instead, enzyme reaction products are concentrated using a tyramide signal amplification system. In our method, tyramide substrate reacts with horseradish peroxidase (HRP) labeled with an immunocomplex on beads, and the substrate is converted into short-lived radical intermediates. By adjusting the bead concentration in the HRP-tyramide reaction and conducting the reaction using freely moving beads, tyramide radicals are deposited only on beads labeled with HRP and there is no diffusion to other beads. Consequently, the fluorescence signal is localized on a portion of the beads, making it possible to count the number of labeled beads digitally. The performance of our method was demonstrated by detecting hepatitis B surface antigen with a limit of detection of 0.09 mIU/mL (139 aM) and a dynamic range of over 4 orders of magnitude. The obtained limit of detection represents a >20-fold higher sensitivity than conventional ELISA. Our method has potential applications in simple in vitro diagnostic systems for detecting ultralow concentrations of protein biomarkers.