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We reported a method to fabricate fluorescent probes preferentially end-linked to Au nanorods. In comparison with organic dyes, the single nanocomplexes provide significant improvement in signal brightness. The bioconjugated nanoantenna structure implicates an approach that is more convenient and amenable to integration for photonic, optoelectronic, and biotechnological applications.
Current biological studies are limited by existing fluorescent probes which suffer from intrinsic deficits, particularly poor photostability and low emission intensities, restraining their applications in dynamics studies1. It is crucial to create a versatile set of probes with improved photostability and higher emission rates to advance studies of individual biomolecule function.
We now know that strong resonant coupling between the excitation light and the noble metal nanoparticles leads to a number of remarkable optical effects, including a considerable near-field enhancement of fluorescence at the metal surface2–4. It suggests that the optical absorption and scattering properties of metallic nanostructures can be used to control the radiative decay rate and direction of fluorophore emission. Enhancement and quenching of fluorescence by noble metal nanoparticles have been reported mainly for spheres, aggregates, and nanoshells5–11. Using the elongated resonance shape and fluorophore distance from the metal surface, the fluorescence can be significantly modified12. We report here the use of single gold nanorods as metallic plasmonic structures. Through covalent linkages of fluorophore at the preferred longitudinal axis of the nanorods, we are able to increase the overall optical signal for improved sensitivity. These effects may be used for increased detectability of single molecules bound to surfaces which contain metallic structures in a nonrandom fashion, for either biophysical studies or high sensitivity assays.
We synthesized gold nanorods in aqueous solution using a seed-mediated route in the presence of cetyltrimethylammonium bromide (CTAB)13. After synthesis, the colloidal suspensions were repeatly centrifuged and washed with deionized water to remove excess surfactant. The average aspect ratio of the rods was 6 (ca. 80 nm in length and 13 nm in diameter) as inferred from transmission electron microscopy (TEM) and the longitudinal plasmon band at 980 nm (supporting information). Functionalization of the nanorods was performed according to the reported method14. Owing to the preferred binding of thiol molecules to the gold nanorod edges rather than the side faces14,15, the biotin-capped gold rod ends was achieved, which led to the end linkage upon subsequent addition of streptavidin (Scheme 1). Aggregation of end-to-end linked Au nanorods was minimized under the selected concentrations. The functionalized nanorods were incubated with a low concentration solution of oligonucleotides labeled with biotin and Cy5 (Biotin-3’-AGG-TGT-ATG-ACC-GGT-AGA-AG-5’-Cy5, ca. 8nm in length) to obtain the hybrid nanocomplexes (supporting information).
Figure 1 shows typical 10×10 µm confocal scanning images taken from the individual Cy5-tagged ssDNA (a) and nanocomposites (b) deposited on glass coverslips, respectively.
The noticeable differences in brightness of emission spots indicate preliminary information of enhanced fluorescence emission in the presence of gold nanostructures. Panel c in Figure 1 illustrates a time trace of a single dye-labeled ssDNA suffered photobleaching as evidence by the discrete drop in fluorescence intensity to the background level. Most of spots investigated suffered permanent photobleaching in an abrupt step, ensuring a single binding event had been observed. Panel b in Figure 1 depicts a typical time trace observed from Au/ssDNA hybrid nanocomplexes. In contrast, much higher emission rate is clearly observed, which is approximately 40-fold greater than that observed in the absence of gold nanostructure. We occasionally noticed two-step or multiple-step photobleaching behaviors (supporting information), showing a possibility that two or more molecules were present under the focusing volume, indicating two or more biotin-ssDNA molecules conjugated to the streptavidin end-capped nanorods. Despite the fact, the overall enhancement in fluorescence intensity was observed for the fluorophores located at the ends of nanorods.
The single molecule fluorescence lifetime measurement is implemented using time-correlated single photon counting by plotting a histogram of time lags between the excitation pulses and the detected fluorescence photons. The analysis of the fluorescence signal of Cy5-ssDNA on glass (Panel d in Figure1) yields an averaged lifetime τ = 2.51 ns. In the contrast, we observed a considerable decrease in lifetime of single Au nanocomplexes (Panel f in Figure1, τ = 0.33 ns). Clearly, the fluorescence decay of the Au nanocomplexes is faster than that of the free probe on glass. This suggests that the fluorescence decay rate of the fluorophore is substantially affected by the Au nanorod.
Figure 2 illustrates brightness histograms acquired from ~80 single molecules of Cy5-ssDNA and Au nanocomplexes, respectively. To aid interpretation, brightness distributions were fit to Gaussian functions. A symmetric distribution with a mean value equals to 8 counts/ms is obtained for dye molecules immobilized on bare glass coverslips, indicating the free probe sample is relatively homogeneous. Brightness of Au hybrid nanocomplexes is shifted well to the much higher values as shown in Figure 2b. In contrast to the symmetric distribution shown in Figure 2a, the brightness distribution is much broad, depicting the likely existence of two different subpopulations of single molecule species arising from diverse near-field electromagnetic interactions. The end-linked dye probes randomly oriented along the longitudinal axis of the nanorods and led to variation of fluorescence enhancements. According to the radiating plasmons (RPs) model, the relatively larger elongated gold nanorods display dominant scattering component over absorption component above 600nm and are expected to enhance fluorescence of fluorophores emitting at wavelengths longer than 600 nm7,16,17. Additionally, we observed the polarization dependence of the excitation, which was demonstrated on a single Au nanocomplex excited at different directions (supporting information). The emission intensity dropped abruptly as the excitation angle rotated 90°, indicating that the magnitude of the enhancement depends on the location of the fluorophore around the particle and the orientation of its dipole moment relative to the metallic surface.
The lifetime histograms of Cy5-ssDNA in the presence and in the absence of Au nanorods illustrate roughly symmetric distributions in two distinct ranges, respectively. Significantly shortened lifetimes of Au nanocomplexes confirm the increased radiative decay rate of the fluorophores in the presence of metallic nano-objective. An increase in the radiative decay rate results in an increased intensity (due to the increased quantum yield) and reduced lifetime17.
In summary, we observed a remarkable increase in fluorescence of single fluorophores controllably linked to ends of Au nanorods. The fluorescence enhancement to nanorod end-linked fluorophore is due to a combination of processes including local electric field intensity contour around the nanorods, modification of the radiative decay rate, and the local orientations of dye molecules. The result suggests a facile approach to controllably fabricate nanometer-scale probes, which is useful for single molecule imaging applications.
This work was supported by NHGRI (Grant HG002655), and NIBIB (Grant EB006521).
SUPPORTING INFORMATION. Experimental details, TEM images, time traces, fluorescence intensity images under polarized excitation lights. Further details are given as noted in the text. This information is available free of charge via the Internet at http://pubs.acs.org.