Biomedical Diagnostics Institute, National Centre for Sensor Research, School of Physical Sciences, Dublin City University, Glasnevin, Dublin 9, Ireland.
Anal Bioanal Chem. 2010 Feb;396(3):1127-34. doi: 10.1007/s00216-009-3357-9. Epub 2009 Dec 13.
In this work, we used a model assay system (polyclonal human IgG-goat antihuman IgG) to elucidate some of the key factors that influence the analytical performance of bioassays that employ metal-enhanced fluorescence (MEF) using silver nanoparticles (NPs). Cy5 dye was used as the fluorescent label, and results were compared with a standard assay performed in the absence of NPs. Two sizes of silver NPs were prepared with respective diameters of 60 +/- 10 and 149 +/- 16 nm. The absorption spectra of the NPs in solution were fitted accurately using Mie theory, and the dipole resonance of the 149-nm NPs in solution was found to match well with the absorption spectrum of Cy5. Such spectral matching is a key factor in optimizing MEF. NPs were deposited uniformly and reproducibly on polyelectrolyte-coated polystyrene substrates. Compared to the standard assay performed without the aid of NPs, significant improvements in sensitivity and in limit of detection (LOD) were obtained for the assay with the 149-nm NPs. An important observation was that the relative enhancement of fluorescence increased as the concentration of antigen increased. The metal-assisted assay data were analyzed using standard statistical methods and yielded a LOD of 0.086 ng/mL for the spectrally matched NPs compared to a value of 5.67 ng/mL obtained for the same assay in the absence of NPs. This improvement of approximately 66x in LOD demonstrates the potential of metal-enhanced fluorescence for improving the analytical performance of bioassays when care is taken to optimize the key determining parameters.
在这项工作中，我们使用了一种模型分析系统（多克隆人 IgG-山羊抗人 IgG）来阐明一些关键因素，这些因素影响使用银纳米粒子 (NPs) 进行生物分析的分析性能。Cy5 染料被用作荧光标记，并将结果与没有 NPs 进行的标准分析进行比较。我们制备了两种大小的银 NPs，分别为 60 ± 10nm 和 149 ± 16nm。使用 Mie 理论准确拟合了 NPs 在溶液中的吸收光谱，并且发现 149nm NPs 在溶液中的偶极共振与 Cy5 的吸收光谱非常匹配。这种光谱匹配是优化 MEF 的关键因素之一。NPs 均匀且可重复地沉积在聚电解质涂覆的聚苯乙烯基底上。与没有 NPs 辅助的标准分析相比，使用 149nm NPs 的分析在灵敏度和检测限 (LOD) 方面均得到了显著提高。一个重要的观察结果是，随着抗原浓度的增加，荧光的相对增强增加。使用标准统计方法对金属辅助分析数据进行了分析，并得到了对于光谱匹配的 NPs 的 LOD 为 0.086ng/mL，而在没有 NPs 的情况下相同分析的 LOD 值为 5.67ng/mL。LOD 提高了约 66 倍，这表明当注意优化关键决定参数时，金属增强荧光有可能提高生物分析的分析性能。
