Increased resonance energy transfer between fluorophores bound to DNA in proximity to metallic silver particles

Abstract

We examined the effects of metallic silver particles on resonance energy transfer (RET) between fluorophores covalently bound to DNA. A coumarin donor and a Cy3 acceptor were positioned at opposite ends of a 23-bp double helical DNA oligomer. In the absence of silver particles the extent of RET is near 9%, consistent with a Forster distance R 0 near 50 Å and a donor to acceptor distance near 75 Å. The transfer efficiency increased when the solution of AMCA-DNA-Cy3 was placed between two quartz plates coated with silver island films to near 64%, as determined by both steadystate and time-resolved measurements. The apparent R 0 in the presence of silver island films increases to about 110 Å. These values of the transfer efficiency and R 0 represent weighted averages for donoracceptor pairs near and distant from the metallic surfaces, so that the values at an optimal distance are likely to be larger. The increased energy transfer is observed only between two sandwiched silvered slides. When we replaced one silvered slide with a quartz plate the effect vanished. Also, the increased energy transfer was not observed for silvered slides separated more than a few micrometers. These results suggest the use of metal-enhanced RET in PCR, hybridization, and other DNA assays, and the possibility of controlling energy transfer by the distance between silver surfaces. Fluorescence resonance energy transfer (RET) 3 is widely used in biochemistry [1][2][3][4] and in DNA biotechnology [5][6][7]. RET is a widely useful phenomenon because the Forster distance (R 0 ) can be readily calculated from the spectral properties of the donor (D) and acceptor (A), and the extent of RET is insensitive to the sample conditions if the sample remains transparent. A disadvantage of RET is the limited distances over which RET occurs. The largest known Forster distances are near 55 Å for organic fluorophores and near 90 Å for lanthanide donors [8][9][10]. In the case of DNA a Forster distance of 50 Å corresponds to approximately 16 bp, so that RET only occurs when the donor and acceptor are closely spaced within the DNA helix. DNA assays based on RET over larger distances have not been developed because longer range RET does not occur.In the present report we describe a new approach which promises to increase the Forster distances by several-fold. At present almost all fluorescent measurements are performed for fluorophores in the free-space conditions, meaning the surrounding medium is mostly homogeneous and transparent to the absorption and emission wavelengths. Under these conditions the observed far-field emission is well described by Maxwell's equations for an *Corresponding author. Fax: 1-410-706-8408. E-mail address: lakowicz@cfs.umbi.umd.edu (J.R. Lakowicz). 1 Current address: Center for Biomolecular Science & Engineering (Code 6900), Naval Research Lab., Washington, DC 20375. 2 Current address: Technical University of Gdansk, Faculty of Applied Physics and Mathematics, ul. Narutowicza 11/12, Poland. 3 Abbreviations used: A, accep...

Distance-Dependent Emission from Dye-Labeled Oligonucleotides on Striped Au/Ag Nanowires: Effect of Secondary Structure and Hybridization Efficiency

“…Lakowicz and co-workers have reported a number of surface effects on fluorescence, including enhanced intrinsic fluorescence from unlabeled DNA, 41 improved photostability, 42 and increased resonant energy transfer between DNA-bound molecular donors and acceptors. 43 This group has also reported increased apparent quantum yields and decreased lifetimes for Cy3-and Cy5-labeled dsDNA adsorbed nonspecifically onto Ag particles. 44 The kinetics of labeled complementary strands to surfacebound probe DNAs have been followed in real time by both surface plasmon field enhanced 21 and epi-illumination 45,46 fluorescence spectroscopy.…”

Single-Molecule Studies of Enhanced Fluorescence on Silver Island Films

“…In the past years we have taken a different approach to create improved probes, this being the use of metallic nanoparticles [1][2][3]. Ensemble experiments have revealed that proximity to silver particles could increase the intensity and photostability of fluorphores [4][5][6][7][8][9]. This effect is due to the through-space nearfield interaction between the fluorphore and surface plasmons on the metals.…”

“…Therefore, we examined the widely used fluorescent probe Cy5 near SIFs. These films consist of sub-wavelength-size silver particles on a glass surface [4,5]. Our single-molecule experiments of this system reveal both the spectral changes due to the metal particles and the heterogeniety due to a range of fluorophore-metal distances.…”

DNA Mediated Energy Transfer from 4',6-Diamidino-2-phenylindole to tetra- and bis-cationic Porphyrins at Low Binding Densities

“…[20][21][22][23][24][25][26][27][28][29] Generally, the emission energy level of a donor molecule coincides with the absorption energy level of the acceptor, in the DNA mediated excited energy transfer, thus, is resonance energy transfer (RET) type in its nature. The excited energy of 4',6-diamidino-2-phenylindole (DAPI, Figure 1) has been reported to transfer to DNA intercalating [Ru (1,10-…”