Ray Krishanu, Zhang Jian, Lakowicz Joseph R
Center for Fluorescence Spectroscopy, University of Maryland at Baltimore, Department of Biochemistry and Molecular Biology, 725 West Lombard Street, Baltimore, MD 21201, USA.
Proc SPIE Int Soc Opt Eng. 2009;7185:71850C. doi: 10.1117/12.808958.
Fluorescence detection is a central component in biological research. In recent years there has been a growing interest in the interactions of fluorophores with metallic surfaces or particles. A single-stranded oligonucleotide was chemically bound to a single 50 nm diameter silver particle and a Cy5-labeled complementary single-stranded oligonucleotide was hybridized with the particle-bound oligonucleotide. The bound Cy5 molecules on the silver particles were spatially separated from the silver surface by the hybridized DNA duplex chains, which were about 8 nm in length, to reduce the competitive quenching. We use fluorescence lifetime correlation spectroscopy (FLCS) with picosecond time-resolved detection to separate the fluorescence correlation spectroscopy (FCS) contributions from fluorophores and metal-conjugated fluorophores. The single Cy5-labeled 50 nm silver particles displayed a factor of 15-fold increase in emission signal and 5-fold decrease in emission lifetimes in solution relative to the Cy5-DNA in the absence of metal. Lifetime measurements support the near-field interaction mechanism between the fluorophore and silver nanoparticle. In this study, FLCS is being applied to a system where the brightness and the fluorescent lifetime of the emitting species are significantly different. Our measurements suggest that FLCS is a powerful method for investigating the metal-fluorophore interaction at the single molecule level and to separate two different species from a mixture solution emitting at the same wavelength. Additionally, the highly bright Cy5-DNA-Ag molecules offer to be excellent probes in high background biological samples.
荧光检测是生物学研究的核心组成部分。近年来，人们对荧光团与金属表面或颗粒之间的相互作用越来越感兴趣。将一条单链寡核苷酸化学连接到单个直径为50纳米的银颗粒上，并使Cy5标记的互补单链寡核苷酸与颗粒结合的寡核苷酸杂交。银颗粒上结合的Cy5分子通过长度约为8纳米的杂交DNA双链链在空间上与银表面分离，以减少竞争性猝灭。我们使用具有皮秒时间分辨检测的荧光寿命相关光谱（FLCS）来区分来自荧光团和金属共轭荧光团的荧光相关光谱（FCS）贡献。相对于没有金属时的Cy5-DNA，单个Cy5标记的50纳米银颗粒在溶液中的发射信号增加了15倍，发射寿命减少了5倍。寿命测量支持了荧光团与银纳米颗粒之间的近场相互作用机制。在本研究中，FLCS被应用于一个发射物种的亮度和荧光寿命显著不同的系统。我们的测量表明，FLCS是一种在单分子水平上研究金属-荧光团相互作用以及从发射相同波长的混合溶液中分离两种不同物种的强大方法。此外，高亮度的Cy5-DNA-Ag分子有望成为高背景生物样品中的优秀探针。
