Homogeneous sandwich immunofluorimetric assays are valued for the rapid, low-cost and accurate detection of analytes in liquid phase. However, their exploitation with analytes covering a wide range of concentrations is limited by low sensitivity and the hook effect. Here, we describe a homogeneous immunofluorimetric system based on the quenching of fluorescence in a Förster resonance energy transfer (FRET) donor/acceptor couple of antibody functionalized with two different dyes, respectively fluorescein (donor) and eosin (acceptor), which form a sandwich multi-component assembly with antibody-functionalized gold nanoparticles (GNPs) in the presence of the analyte. The resulting cooperative fluorescence quenching is assisted by the GNPs scaffold through the nanomaterial-surface energy transfer (NSET) effect, which gives an extended linear response versus the antigen concentration that is not possible with the bi-component assays. This immunofluorimetric method allows accurate, reproducible and immediate detection of C-reactive protein (CRP) in the wide concentration range of clinical interest (over two orders of magnitude from 3.5 to 455 nM, 0.4–52 mg/L), without the hook effect. Moreover, the method does not require sample treatment or washing steps. The concept of this multi-component FRET/NSET fluorescence quenching system can be extended to any analyte amenable to the detection with homogeneous sandwich assays.

均相夹心免疫荧光测定法可用于快速，低成本和准确地检测液相中的分析物。但是，由于灵敏度低和钩效应，限制了它们使用涵盖广泛浓度范围的分析物的开发。在这里，我们描述了一种均质免疫荧光系统，该系统基于Förster共振能量转移（FRET）供体/受体对中的荧光淬灭，该抗体用两种不同的染料功能化，分别是荧光素（供体）和曙红（受体），形成三明治在分析物存在下使用抗体功能化的金纳米颗粒（GNP）进行多组分组装。GNP支架通过纳米材料-表面能量转移（NSET）效应协助产生协同荧光猝灭，相对于抗原浓度，线性响应相对于双组分检测无法实现。这种免疫荧光方法可在临床上很宽的浓度范围内（从3.5到455 nM，0.4-52 mg / L超过两个数量级）准确，可重现和立即检测C反应蛋白（CRP），而没有钩效应。而且，该方法不需要样品处理或洗涤步骤。这种多组分FRET / NSET荧光猝灭系统的概念可以扩展到适用于均质夹心检测的任何分析物。可在广泛的临床关注浓度范围内（3.5到455 nM，0.4–52 mg / L的两个数量级）重现性和即时检测C反应蛋白（CRP），而没有钩效应。而且，该方法不需要样品处理或洗涤步骤。这种多组分FRET / NSET荧光猝灭系统的概念可以扩展到适用于均质夹心检测的任何分析物。可在广泛的临床关注浓度范围内（3.5到455 nM，0.4–52 mg / L的两个数量级）重现性和即时检测C反应蛋白（CRP），而没有钩效应。而且，该方法不需要样品处理或洗涤步骤。这种多组分FRET / NSET荧光猝灭系统的概念可以扩展到适用于均质夹心检测的任何分析物。