Directional surface plasmon-coupled emission: application for an immunoassay in whole blood.
Directional surface plasmon-coupled emission: application for an immunoassay in whole blood.
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DOI：
10.1016/j.ab.2005.07.005
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发表时间：
2005-09
影响因子：
2.9
通讯作者：
E. Matveeva;Z. Gryczynski;J. Malicka;Joanna Lukomska;S. Makowiec;K. Berndt;J. Lakowicz;I. Gryczynski
中科院分区：
文献类型：
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作者：
E. Matveeva;Z. Gryczynski;J. Malicka;Joanna Lukomska;S. Makowiec;K. Berndt;J. Lakowicz;I. Gryczynski
我们提出了一种新的方法进行荧光免疫测定全血荧光标记的抗兔免疫球蛋白G（IgG）的银表面。这种基于表面等离子体激元耦合发射（SPCE）的方法由于从位于生物亲和表面附近的荧光团中排他性地选择信号而提供了增加的灵敏度和显著的背景降低。本文描述了一个光学致密的样品基质，即人全血和血清，对SPCE的强度的影响。将抗原（兔IgG）吸附到覆盖有薄银金属层的载玻片上，并检测来自荧光团标记的抗兔抗体的SPCE信号，所述抗体结合到固定的抗原上。研究了样品基质（缓冲液、人血清或人全血）对终点免疫测定SPCE信号的影响。结果表明，结合动力学可以直接在全血或血清中监测。结果表明，与缓冲液相比，人血清和人全血分别仅使SPCE终点信号和免疫测定动力学信号衰减约2倍和3倍，从而产生即使在全血中也容易检测到的信号。血红蛋白的高光吸收是可以容忍的，因为只有距离金属膜几百纳米内的荧光团才有助于SPCE。200 nm层外的激发荧光团对SPCE没有贡献，并且它们的自由空间发射不会通过不透明金属膜透射到玻璃基板中。我们相信，SPCE有可能成为一种强大的方法，用于直接在致密样品（如全血）中进行基于表面结合分析物或许多生物标志物抗体的免疫测定，而无需洗涤步骤。
We present a new approach for performing fluorescence immunoassay in whole blood using fluorescently labeled anti-rabbit immunoglobulin G (IgG) on a silver surface. This approach, which is based on surface plasmon-coupled emission (SPCE), provides increased sensitivity and substantial background reduction due to exclusive selection of the signal from the fluorophores located near a bioaffinity surface. This article describes the effect of an optically dense sample matrix, namely human whole blood and serum, on the intensity of the SPCE. An antigen (rabbit IgG) was adsorbed to a slide covered with a thin silver metal layer, and the SPCE signal from the fluorophore-labeled anti-rabbit antibody, binding to the immobilized antigen, was detected. The effect of the sample matrix (buffer, human serum, or human whole blood) on the end-point immunoassay SPCE signal was studied. It was demonstrated that the kinetics of binding could be monitored directly in whole blood or serum. The results showed that human serum and human whole blood attenuate the SPCE end-point signal and the immunoassay kinetic signal only approximately two- and threefold, respectively, as compared with buffer, resulting in signals that are easily detectable even in whole blood. The high optical absorption of the hemoglobin can be tolerated because only fluorophores within a couple of hundred nanometers from the metallic film contribute to SPCE. Excited fluorophores outside the 200-nm layer do not contribute to SPCE, and their free space emission is not transmitted through the opaque metallic film into the glass substrate. We believe that SPCE has the potential of becoming a powerful approach for performing immunoassays based on surface-bound analytes or antibodies for many biomarkers directly in dense samples such as whole blood with no need for washing steps.