KU Leuven , Laboratory of Solid-State Physics and Magnetism , Celestijnenlaan 200D , B-3001 Leuven , Belgium.
imec , Kapeldreef 75 , 3001 Heverlee , Belgium.
Anal Chem. 2018 Apr 3;90(7):4263-4267. doi: 10.1021/acs.analchem.7b04822. Epub 2018 Mar 15.
Most fluorescent immunoassays require a wash step prior to read-out due to the otherwise overwhelming signal of the large number of unbound (bulk) fluorescent molecules that dominate over the signal from the molecules of interest, usually bound to a substrate. Supercritical angle fluorescence (SAF) sensing is one of the most promising alternatives to total internal reflection fluorescence for fluorescence imaging and sensing. However, detailed experimental investigation of the influence of collection angle on the SAF surface sensitivity, i.e., signal to background ratio (SBR), is still lacking. In this Letter, we present a novel technique that allows to discriminate the emission patterns of free and bound fluorophores simultaneously by collecting both angular and spectral information. The spectrum was probed at multiple positions in the back focal plane using a multimode fiber connected to a spectrometer and the difference in intensity between two fluorophores was used to calculate the SBR. Our study clearly reveals that increasing the angle of SAF collection enhances the surface sensitivity, albeit at the cost of decreased signal intensity. Furthermore, our findings are fully supported by full-field 3D simulations.
大多数荧光免疫分析由于大量未结合（大量）荧光分子的信号过于强烈，在读取之前需要进行洗涤步骤，这些分子会超过通常与基质结合的感兴趣分子的信号。超临界角荧光（SAF）感测是替代全内反射荧光进行荧光成像和感测的最有前途的方法之一。然而，对于收集角度对 SAF 表面灵敏度（即信号与背景比（SBR））的影响，仍然缺乏详细的实验研究。在这封信中，我们提出了一种新的技术，通过同时收集角和光谱信息来同时区分游离和结合荧光团的发射模式。使用连接到光谱仪的多模光纤在背焦平面的多个位置探测光谱，并使用两种荧光团之间的强度差异来计算 SBR。我们的研究清楚地表明，增加 SAF 收集角度可以提高表面灵敏度，但代价是信号强度降低。此外，我们的发现得到了全场 3D 模拟的充分支持。
