Sabanayagam Chandran R, Lakowicz Joseph R
Department of Biochemistry and Molecular Biology, Center for Fluorescence Spectroscopy, University of Maryland School of Medicine, 725 West Lombard Street, Baltimore, MD 21201, USA.
Nucleic Acids Res. 2007;35(2):e13. doi: 10.1093/nar/gkl1054. Epub 2006 Dec 14.
The effects of metal-enhanced fluorescence (MEF) have been measured for two dyes commonly used in DNA microarrays, Cy3 and Cy5. Silver island films (SIFs) grown on glass microscope slides were used as substrates for MEF DNA arrays. We examined MEF by spotting biotinylated, singly-labeled 23 bp DNAs onto avidin-coated SIF substrates. The fluorescence enhancement was found to be dependent on the DNA spotting concentration: below approximately 12.5 muM, MEF increased linearly, and at higher concentrations MEF remained at a constant maximum of 28-fold for Cy5 and 4-fold for Cy3, compared to avidin-coated glass substrates. Hybridization of singly-labeled oligonucleotides to arrayed single-stranded probes showed lower maximal MEF factors of 10-fold for Cy5 and 2.5-fold for Cy3, because of the smaller amount of immobilized fluorophores as a result of reduced surface hybridization efficiencies. We discuss how MEF can be used to increase the sensitivity of DNA arrays, especially for far red emitting fluorophores like Cy5, without significantly altering current microarray protocols.
已对DNA微阵列中常用的两种染料Cy3和Cy5测量了金属增强荧光（MEF）的效果。在玻璃显微镜载玻片上生长的银岛膜（SIFs）用作MEF DNA阵列的底物。我们通过将生物素化的、单标记的23 bp DNA点样到抗生物素蛋白包被的SIF底物上来检测MEF。发现荧光增强取决于DNA点样浓度：在约12.5 μM以下，MEF呈线性增加，而在较高浓度下，与抗生物素蛋白包被的玻璃底物相比，Cy5的MEF保持在恒定最大值28倍，Cy3为4倍。单标记寡核苷酸与阵列单链探针的杂交显示，Cy5的最大MEF因子较低，为10倍，Cy3为2.5倍，这是由于表面杂交效率降低导致固定荧光团的量较少。我们讨论了如何利用MEF提高DNA阵列的灵敏度，特别是对于像Cy5这样发射远红光的荧光团，而无需显著改变当前的微阵列协议。
