Single-Molecule Studies of Enhanced Fluorescence on Silver Island Films

Abstract

We investigated enhanced fluorescence of Cyanine 5 dyesnear silver island films (SIFs) by the use of single moleculespectroscopic method. We found that, on average, the Cy5molecules are 18-fold brighter on SIFs than on a glasssurface and that single-molecule lifetimes are 10-foldshorter on SIFs as compared to glass. We observed Cy5molecules until photobleaching occurred and found that themolecules on the SIF emitted 20-fold more photons ascompared to those on glass prior to photobleaching. Theseresults demonstrate that the use of fluorophore–metalinteractions can increase the brightness and photostabilityof fluorophores for single-molecule detection.During the past decade, there has been considerableprogress in the development of fluorescence probes. Manyprobes, such as the cyanine dyes, have high extinctioncoeffiecients and high quantum yields. Hence, it is unlikelythat the future probe development will result in dramaticincreases in detectability for small organic fluorphores. Forthis reason, there is an interest in quantum dots andpolymeric probes. In the past years we have taken adifferent approach to create improved probes, this being theuse of metallic nanoparticles [1–3]. Ensemble experimentshaverevealedthatproximitytosilverparticlescouldincreasethe intensity and photostability of fluorphores [4–9]. Thiseffect is due to the through-space near-field interactionbetween the fluorphore and surface plasmons on the metals.Thus, the use of metallic nanostructure to enhancefluorescence has great potential for applications in thefields of medical diagnostics and biotechnology.The fluorescence spectral properties of fluorphores areespecially important for sensitive detection. The photo-stability of the fluorphore resolves the time it can beobserved prior to photobleaching. Hence, it is of interest todetermine if the improved spectral properties observed atthe ensemble level extends to the single-molecule level.The interactions of fluorophores with metallic particleshave been studied theoretically and empirically at thesingle-molecule level in recent years [10–13]. In thesereports, the major concern was the dependence of theemission rate on the distance between the dye layer and themetallic surface. The emission can be quenched due toradiation energy transfer to the metal as molecules adsorbeddirectly on the surface [12, 13]. Another consideration is theenhanced fluorescence due to increased electromagneticfield of surface plasmon and/or enhanced quantum yield[10, 11]. Therefore, we examined the widely used fluores-cent 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 boththe spectral changes due to the metal particles and theheterogeniety due to a range of fluorophore–metaldistances.For these studies, the Cy5 molecules were spin-coatedonto the SIFs from an aqueous solution containing 0.5%polyvinyl alcohol (PVA). Initially, we examined theensemble emission spectra in which the Cy5 concentrationis 20-fold higher than for the single-molecule experiments(Figure 1). Both Cy5 spectra exhibit emission maxima near680 nm. A significant enhancement in fluorescenceemission is clearly evident on silvered surfaces. Thepresence of silver nanostructure did not distort the Cy5spectrum. Additionally, the SIF sample without Cy5 did notcontribute significant background. This initial ensembleresult indicates that the Cy5 dyes deposited on SIF display