Surface roughness is known to provide a mechanism for coupling incident light to surface plasmons and of creation of far-field light from the plasmons [28
]. For this reason we used a slow gold deposition rate to result in a rough metal surface [6
]. All gold films in the current experiment were grown at a relatively slow rate of 0.1 nm/10 s under a pressure of ca
. 5 × 10−7
]. The surface morphology was controlled principally by the film thickness (2 – 100 nm). A thin Au film (2 nm) displayed a plasmon absorbance at 600 nm (), close to the wavelength of nano-size individual particle, indicating that the thin film was indeed composed of nano-size particles [30
]. The plasmon wavelength was red-shifted to 680 nm with increasing the thickness to 5 nm, showing that the particle size became larger for the thicker gold film (). In the other words, the size of particle increased with the thickness of Au film. When the film thickness was over 10 nm, the plasmon absorbance disappeared, inferring that the metal film became continuous without the presence of individual particles.
Absorbance spectra of Au films of varying thickness, 2 nm, 5 nm, and 10 nm, deposited at 0.1 nm/10 s on glass substrate.
The change of surface morphology with the thickness of gold film was characterized by AFM. A thin gold film (2 nm) was found to be composed of individual particles . The average height was ca. 3 nm. Considering that the particles were packed separately on the solid substrate, this height was plausible for an average thickness of 2 nm measured by microbalance. With an increasing thickness of the metal film, it was shown that the particle size became larger. If the gold film was further increased, the individual particles were aggregated and the metal film became more and more continuous . While this AFM image indicates the surface consists of separated particles the extinction spectrum in suggests the film is continuous or that the particles are strongly interacting. The height was ca. 14 nm, close to the thickness measured by the microbalance.
AFM planar and 3D images of 2 nm Au film, 10 nm Au film, and 10 nm Au film coated by 10 nm silica.
In order to avoid a strong quenching due to the close proximity of fluorophores to the metal surface, and to protect the metal film, a silica layer was coated on the metal by the vapor deposition method in the same chamber [30
]. Because a portion of silicon monoxide was oxidized to silicon dioxide by oxygen left in the chamber during the evaporation, the silica layer was supposed to be composed of silicon monoxide and dioxide mixture. The AFM image of 10 nm silica-coated gold film showed smooth regions different from the metal film, although there were obvious regions of roughness . The average
height of these defects was ca
. 10 nm, which was consistent with the thickness of silica measured by microbalance.
We studied the effects of the gold surfaces and the thickness of the gold surfaces on the emission spectra of the AlexaFluor-555 labeled antibody (Alexa-IgG). All gold films were coated by 5 nm thick silica. Rabbit IgG was adsorbed by physiosorption on the silica [26
], and Alexa Fluor-555 anti Rabbit IgG was bound to the immobilized IgG. The concentration of bound fluorophore could be estimated quantitatively through the luminescence intensity change in buffer solution before and after binding. Because there were average
4.5 fluorophores on each antibody molecule, the antibody coverage on the silica was estimated to be ca
. 1 × 10−12
. It was known that one IgG molecule could bind 1 – 2 antibody molecules, so the coverage of IgG molecule was inferred to be less than 2 × 10−12
. This coverage was found to be almost independent of the thickness of gold and silica.
Emission spectra are shown in . The fluorescence intensity on the 2 nm gold film was close to that on the glass substrate. The quenching effects of gold were not seen, possibly as a result of the 5 nm layer of silica. The intensity of the labeled IgG increase with increasing mass thickness of gold, reaching saturation above 30 nm (, insert). Remarkably, instead of quenching, the enhancement reached 6-fold for an approximate thickness of 30 nm. This enhancement is less than that reported silver, but the enhancement is substantial.
Fig. 3 Emission spectra of Alexa Fluor-555 labeled anti-Rabbit IgG on varying thickness gold films upon excitation at 514 nm. The inset represents the dependence of the enhancement factor on the thickness of gold film. All gold films were coated by 5 nm silica. (more ...)
In this experiment, besides the thickness of silica layer, the thickness of IgG has to be considered. The IgG molecule can be described as a cylinder shape with a 4 nm diameter and a 10 nm height. So the thickness of protein layers is about 5 – 10 nm when it was adsorbed on the solid substrate vertically and horizontally. It means that there exists a spacer between the dye-labeled antibody and metal surface. In addition, because the thin metal film is not continuous, some fluorophores are adsorbed on the glass instead of the metal island. Hence, even though the protein layer is adsorbed directly on the metal without a separation by the silica, the fluorophores are not quenched. It is of interest to estimate the optimal distance for MEF. Combining the thickness of silica and the proteins, the distance of maximum MEF was about 15 – 20 nm. This distance seems to be slightly longer than that found optimal for MEF with silver, which is typically near 10 nm. At this time the data are not adequate to state with certainly the gold MEF occurs at larger distances than silver MEF, but this result seems reasonable given the quenching effect of gold at short distances.
It is interesting to know the role of localized surface plasmon (LSP) resonances or surface plasmon polariton (SPP) resonance in the observed fluorescence enhancement. There is no complete answer to this question because there is a continuous transition from LSP to SPP with increasing the metal thickness. Even with the imperfect separation of LSP and SPP, we believe the dominated cause of enhancement on LSP. This is because our samples remain rough even at the large gold thickness. The samples are not illuminated under the condition when SPP can be created on a smooth surface, so the created plasmon is mostly LSP for all the metal thickness.
An important property of metal-enhanced fluorescence is a reduction in lifetimes occurring simultaneously with increases in intensity. We measured the time-dependent decays of the surface-bound protein. We used the frequency-domain (FD) method. The FD data were analyzed in terms of the multi-exponential model [31
are the amplitudes and τi
the decay times, Σ αi
= 1.0. The amplitude-weighted lifetime is given by:
The contribution of each decay component to the steady state intensity is given by
The average lifetime is given by
The values of αι and τi
were determined by non-linear least squares impulse reconvolution with a goodness-of-fit χ2R
criterion. After fitting to the multiexponential model, the recovered parameters and lifetimes were listed in . We believe the shorter lifetimes are due to an increase in the radiative decay rate, but we cannot rule out quenching of some portion of the population. The amplitude-weighted lifetime displayed a tendency of increase with the thickness of the gold film. The lifetime on 10 nm silica was shorter than that on 5 nm silica when the thickness of gold film was 10 nm, indicating that the intrinsic decay rate became faster when the fluorophore was localized at a certain distance from the gold surface. The recovered intensity decays for Alexa 555-IgG from the parameters in were plotted in . The intensity decays on 5, 10, and 20 nm thick gold films are more rapid than on glass. Short decay time components are seen on the 5 and 10 nm thick films, as expected for fluorophores near metal particles [5
]. The short lifetime component is less prominent on the 20 nm film and the intensity decay becomes comparable to glass on the 50 nm film. The smaller changes in lifetimes means the thicker metal film is in agreement with recent theory for a fluorophore above a smooth metal film and with our own unpublished observations.
Lifetime data obtained using the multi-exponential model for the fluorophore coated on the glass substrate or metal surface.
Recovered intensity decays for Alexa 555-IgG from the parameters in .
We also bound Alexa Fluor-680 anti-Rabbit IgG to the protein-coated surface with 5 nm silica. The fluorescence spectra of Alexa Fluor-680 displayed the maximum at 698 nm upon excitation at 610 nm (). The intensity was observed to increase more greatly than that of Alexa Fluor-555 under the same conditions. The maximum enhancement of Alexa Fluor-680 is about 10 fold (inset of ), higher than that of Alexa Fluor-555 indicating that the near IR dye can be enhanced more efficiently on the Au film. This result consists with our previous observation on the silver [30
Emission spectra of Alexa Fluor-680 labeled anti-Rabbit IgG on a 50 nm gold film upon excitation at 610 nm. The inset represents the dependence of the enhancement factor on the thickness of gold film. All gold films were coated by 5 nm silica.