We examined the silver electrodes during passage of electrical current. Upon starting the current, we observed rapid growth of fractal-like structure on the cathode (). These structures grew rapidly and appeared to twist as they grew. After a short time the silver began to grow onto the glass slide (). These structures are similar to those reported recently during electroplating of insulators.17
We found that dipping the slides in 0.001 mg/dL of SnCl2
for 30 min prior to electrolysis resulted in structures which were firmly bound to the glass during working. Without SnCl2
similar nanostructures were formed but were partially removed during washing.
Silver nanostructures on, or near, the silver cathode treated with SnCl2. Bright field image.
Silver nanostructures deposited on glass during electroplating (A). Panels B and C are consecutive magnifications of the marked area on panel A. Bright field image.
We examined the usefulness of these silver structures for metal-enhanced fluorescence (MEF). Following passage of current, the silvered slide was soaked in 10 µM FITC-HAS overnight at 4 °C, which is thought to result in a monolayer of surface-bound HSA.21,24
This sample of FITC-HSA contained approximately seven fluorescein molecules per HSA molecule, so that the fluorescein emission was partially quenched by resonance energy transfer (RET) between the fluorophores.21
Using partially self-quenched fluorescein also potentially affords for the “release” or “unquenching” of fluorescence, as the fluorescein is already self-quenched. These over-labeled proteins are ideal choices as probes, because the modified radiative decay rate near the metallic silver surface competes with quenching (a nonradiative process), in essence returning the quenched fluorescence signal. While our probe is somewhat exotic in its unquenching nature near silver, a similar effect is also observed for a single label, but at a reduced total intensity.
We measured a fluorescence image of a fractal silver surface using the same apparatus () with 442-nm excitation (). The fluorescence image of the area in shows a fluorescence pattern which closely follows the shape of the silver particles (). Interestingly, regions of high and low fluorescence intensity were observed. This result is roughly consistent with recent SERS data which showed the presence of intense signals, which appeared to be located between clusters of particles.25–26
Emission spectra were collected from eight selected regions of varying brightness. In all cases, the emission spectra appeared to be that of fluorescein (). In these spectra the blue edge of the fluorescein emission is cut off by the emission filter. As a blank control, the silver structures were coated with unlabeled HSA. The resulting signal was lower than those of any of the silvered areas and lower than regions of the unsilvered glass treated with FITC-HSA.
Fluorescence image of FITC-HSA deposited on the silver structure shown in .
Emission spectra of the numbered areas marked in .
We spatially scanned the fluorescence emission intensity on a diagonal path across the silver structure (). The emission intensity varied roughly with the density of the deposited silver (). The lower dotted line in represents the intensity of FITC-HSA on unsilvered glass. Most of this signal appears to be due to dark counts in the CCD camera. The emission intensities range from 100-fold (position 6) to 600-fold (position 1) greater than the signal from FITC-HSA on unsilvered glass. We recognized that some of this increase could be due to binding of more FITC-HSA to silver structures with large surface areas. We note that the fluorescein is not quenched on the surface, probably because the size of an HSA molecule positions the fluorescein about 40 Å from the surface, which is near the distance for maximal radiative rate and therefore fluorescence enhancement.27
Diagonal scan of the emission intensity of FITC-HSA. The dashed line is the intensity observed across a line of equivalent length across unsilvered glass.
There are probably several contributions to the increased intensities of FITC-HSA on the fractal silver surfaces. These causes include more protein binding, increased rates of excitation due to an enhanced electric field, and larger quantum yields due to increased rates of radiative decay. If the radiative decay rate is increased, then the lifetime should decrease.9
We measured the frequency-domain intensity decays of FITC-HAS bound to unsilvered glass and fractal silver (). The amplitude-weighted lifetime of FITC-HSA bound to glass is about 80 ps, in agreement with previous measurements of self-quenched fluorescein on HSA.21
On fractal silver the amplitude-weighted lifetime is dramatically reduced to about 3 ps. We carefully considered whether this decrease was due to detection of scattered light. The background signal from unlabeled HAS on fractal silver was less than 1%, too quick to be resolved by our instrumentation. The emission filter combination of a 540-nm interference filter and a solution of CrO42−
was selected for low emission from the filter when exposed to scattered light from the sample. We believe part of the increased intensity is due to release of fluorescein self-quenching due to the silver surface.21
Frequency-domain intensity decays of FITC-HSA on glass (top) and deposited silver (bottom).
We tested the use of fractal silver for providing emission selectively from fluorophores in close proximity to the surface. For this purpose we examined glass coated with FITC-HSA, to which we added a solution of nile blue, to yield an over 10-fold larger signal from nile blue (, top). In contrast to the glass surface, for the silver surface the signal was now dominated by the FITC-HSA (bottom). Intuitively, this result suggests that unwanted autofluorescence from biological samples can be avoided by metal-enhance fluorescence of surface-localized fluorophores.
Emission spectra of a monolayer of FITC-HSA on glass (top) or silver (bottom) in the presence of 2 × 10−6 M nile blue. The sample was 100 µM thick.
And finally, we studied the photostabilities of FITC on the fractal silver surface, silver island films (SIFs), and uncoated quartz. While the relative photobleaching is higher on fractal silver, the increased rate of photobleaching is less than the increase in intensity (). From the areas under these curves we estimate 16- and 160-fold more photons can be detected from FITC-HSA on SIFs or fractal silver, respectively, relative to quartz, prior to photobleaching.
Photostability of FITC-HSA deposited on glass (•••), SIF (– – –), and fractal silver (−). The samples were illuminated at 514 nm.