Silver-Gold Nanocomposite Substrates for Metal-Enhanced Fluorescence: Ensemble and Single-Molecule Spectroscopic Studies

Abstract

In recent years, there has been a growing interest in the studies involving the interactions of fluorophores with plasmonic nanostructures or nanoparticles. These interactions lead to several favorable effects such as increase in the fluorescence intensities, increased photostabilities, and reduced excited-state lifetimes that can be exploited to improve the capabilities of present fluorescence methodologies. In this regard, we report the use of newly developed silver-gold nanocomposite (Ag-Au-NC) structures as substrates for metal-enhanced fluorescence (MEF). The Ag-Au-NC substrates have been prepared by a one-step galvanic replacement reaction from thin silver films coated on glass slides. This approach is simple and suitable for the fabrication of MEF substrates with large area. We have observed about 15-fold enhancement in the fluorescence intensity of ATTO655 from ensemble fluorescence measurements using these substrates. The fluorescence enhancement on the Ag-Au-NC substrates is also accompanied by a reduction in the fluorescence lifetime of ATTO655, which is consistent with the fluorophore-plasmon coupling mechanism. Single-molecule fluorescence measurements have been performed to gain more insight into the metal-fluorophore interactions and to unravel the heterogeneity in the interaction of individual fluorophores with the fabricated substrates. The single-molecule studies are in good agreement with the ensemble measurements and show maximum enhancements of ~50-fold for molecules located in proximity to the "hotspots" on the substrates. In essence, the Ag-Au-NC substrates have a very good potential for various MEF applications.

Figure 1

SEM images of the thermally evaporated Ag film before (A) and after (B) reaction with HAuCl₄ for 15 min and their EDS spectra (C).

Figure 2

AFM image (10 μm × 10 μm) and a line profile across the Ag–Au–NC substrate.

Figure 3

(A) Fluorescence emission spectra of ATTO655-SA on glass, the thermally evaporated Ag film before the galvanic reaction (s) and the Ag–Au–NC substrate. (B) Fluorescence decay traces of ATTO655-SA on glass and Ag–Au–NC substrate.

Figure 4

Scanning confocal images (20 μm × 20 μm) of ATTO655-SA on (A) glass and (B) Ag–Au–NC substrate. Scale bars show the respective intensity counts in 0.6 ms bin.

Figure 5

Intensity–time trajectories of individual ATTO655-SA molecules on (A) glass and (B) Ag–Au–NC substrate.

Figure 6

(A) Single-molecule fluorescence spectra of individual ATTO655-SA on glass and Ag–Au–NC substrate. (B) Fluorescence intensity decays of individual ATTO655-SA on glass and Ag–Au–NC substrate.

Figure 7

Fluorescence intensity (count rate) histogram of ATTO655-SA on (A) glass and (B) Ag–Au–NC substrate. Average fluorescence lifetime histogram of ATTO655-SA on (C) glass and (D) Ag–Au–NC substrate. The histograms have been constructed for ~70 single molecules in each case.

Scheme 1

Schematic Representation of Ag–Au–NC Substrate Fabrication (A) and the Immobilization of ATTO655 on (B) Glass, (C) Ag Film, and (D) Ag–Au–NC Substrates through the BSA-Biotin-Streptavidin Chemistry