Hybridization chain reaction-based fluorescence immunoassay using DNA intercalating dye for signal readout.
Hybridization chain reaction-based fluorescence immunoassay using DNA intercalating dye for signal readout.
复制标题
DOI：
10.1039/c4an00190g
复制
发表时间：
2014-06
期刊：
影响因子：
--
通讯作者：
中科院分区：
文献类型：
--
作者：
基于杂交链反应（HCR）技术，构建了一种新型的荧光免疫吸附分析方法，该方法采用DNA嵌入染料作为信号读出器，可在竞争和夹心两种模式下对目标进行灵敏检测。在该平台中，捕获和识别过程基于免疫反应，信号放大取决于无酶、等温HCR诱导的标记事件。在竞争性或夹心免疫反应后，生物素化的捕获DNA通过抗生物素蛋白与生物素化的信号抗体结合，并通过两个特异性发夹将HCR触发成带切口的双螺旋。将双链DNA荧光探针基因荧光素（GF）嵌入扩增的DNA链中，产生荧光信号。HCR/GF免疫分析法在竞争模式下对兔IgG的检测限（LOD）比传统的荧光免疫分析法（以异硫氰酸荧光素标记的链霉亲和素或异硫氰酸荧光素标记的第二抗体作为信号读出）提高了100倍以上。以甲胎蛋白为模型靶点，采用夹心法建立了甲胎蛋白荧光免疫分析方法，线性范围为28 ng mL（-1）~ 20 μg mL（-1），检出限为6.0 ng mL（-1）。该方法在加标人血清中也显示出令人满意的分析，这表明它可能在生命科学和即时诊断中具有广泛的应用潜力。
A novel format of fluorescence immunosorbent assay based on the hybridization chain reaction (HCR) using a DNA intercalating dye for signal readout was constructed for the sensitive detection of targets, both in competitive and sandwich modes. In this platform, the capture and recognition processes are based on immunoreactions and the signal amplification depends on the enzyme-free, isothermal HCR-induced labelling event. After a competitive or a sandwich immunoreaction, a biotinylated capture DNA was bound to a biotinylated signal antibody through avidin, and triggered the HCR by two specific hairpins into a nicked double helix. Gene Finder (GF), a fluorescent probe for double-strand DNA, was intercalated in situ into the amplified chain to produce the fluorescence signal. The limit of detection (LOD) for rabbit IgG in competitive mode by HCR/GF immunoassay was improved at least 100-fold compared with the traditional fluorescence immunoassay using the fluorescein isothiocyanate-labelled-streptavidin or fluorescein isothiocyanate-labelled second antibody as the signal readout. The proposed fluorescence immunoassay was also demonstrated by using α-fetoprotein as the model target in sandwich mode, and showed a wide linear range from 28 ng mL(-1) to 20 μg mL(-1) with a LOD of 6.0 ng mL(-1). This method also showed satisfactory analysis in spiked human serum, which suggested that it might have great potential for versatile applications in life science and point-of-care diagnostics.