Abstract
Due to the energy transfer from surface plasmons, the fluorescence of fluorophores near metallic nanostructures can be enhanced. This effect has been intensively studied recently for biosensor applications. This work reports on the luminescence enhancement of 100 nm Cy3 dye-doped polystyrene nanoparticles by energy transfer from surface plasmons of gold colloidal nanoparticles with sizes of 20 and 100 nm. Optimal luminescence enhancement of the fluorophores has been observed in the mixture with 20 nm gold nanoparticles. This can be attributed to the resonance energy transfer from gold nanoparticles to the fluorophore beads. The interaction between the fluorophores and gold particles is attributed to far-field interaction.
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1. Introduction
Surface plasmons (SPs) are coupled modes of electromagnetic field and free electrons in metal. The interaction of fluorescent molecules with plasmons in metallic nanostructures and the physical phenomena related to nanoscale confinement of light have attracted the interest of physicists over recent years [1, 2]. Metallic nanostructures can enhance the fluorescence of nearby fluorophores because the interactions between the dipole moments of the fluorophores and the surface plasmon field of the metal can increase the incident light field, which results in enhanced local fluorescence intensity and rate of excitation [3–6]. Lakowicz [4] reported that the proximity of fluorophores within about 10 nm of silver island films resulted in increased emission intensities and decreased lifetimes. Similar enhancement effects were also observed with silver colloids, fractal silver surfaces, thin silver or gold films or gold colloidal nanoparticles. For example, enhancement of the fluorescence intensity of fluorophore dye (Rose Bengal, FITC-HAS, etc.) was observed on a quartz plate or a glass substrate with gold nanoparticles (GNPs) immobilized on it [7, 8]. Numerous reports on theoretical and experimental studies show that the morphology and size of metal particles affect the resonance frequency of surface plasmons and the enhancement factor of a local field that can enhance fluorescence [4–9]. A radiating plasmons model is used [4] to determine when a particular metal structure will quench or enhance fluorescence. The emission enhancement or quenching of a fluorophore near the metal can be predicted from the optical properties of the metal structures as calculated from electrodynamics, Mie theory or Maxwell's equations. For example, according to Mie theory and the size and shape of the particle, the extinction of metal colloids can be due to either absorption or scattering. If the absorption process is dominant over scattering, the fluorescence is quenched, but fluorescence enhancement occurs when the scattering is dominant. The interactions of fluorophores with plasmons in metallic particles and surfaces have been used in many applications such as obtaining increased fluorescence intensities, developing assays based on fluorescence quenching by gold colloids, or obtaining directional radiation from fluorophores near thin metal films [4–6].
In Drexhage reflective-field conditions fluorescent enhancement can take place in fluorophores at a distance of tens to hundreds of nanometers due to the surface-plasmon-coupled emission [3]. In this work, we study the luminescence enhancement of 100 nm Cy3 dye-doped polystyrene nanoparticles caused by energy transfer from surface plasmons of GNPs and observe that luminescence enhancement can occur at further distances (about a micrometer in solution) from metal particles. Optimal luminescence enhancement of the fluorophores has been observed in the mixture containing GNPs with a size of 20 nm. This can be attributed to the resonance energy transfer from gold GNPs to the fluorophore beads. The interaction between the fluorophores and GNPs is attributed to far-field interaction.
The 100 nm Cy3 dye-doped polystyrene nanoparticles (Cy3 DDPNPs) used in this work are orange fluorescent 100 nm carboxylate-modified FluoSpheres ® beads (Invitrogent), which have an absorption peak at 540 nm and emission peak at 560 nm. These nanoparticles have many biological applications such as super-resolution imaging of cells, analysis of interaction between biomolecules and fluorescence laser tracking [10–12]. Gold colloidal nanoparticles were added to Cy3 DDPNP solution to investigate the luminescence intensity affected by GNPs. We found that the enhancement factor strongly depends on the size of the gold particles, and maximum enhancement is observed when GNPs have a diameter of 20 nm.
2. Experimental
The 100 nm Cy3 dye-doped polystyrene nanoparticles (DDPNPs) were purchased from Invitrogent (CA, USA). These beads have pendent carboxylic acids, making them suitable for covalent coupling of proteins and other amine-containing biomolecules [10–12]. The concentration of the studied beads in the mother solution is 3.64×1014(ml)-1. The bead solution is diluted 104 times. The fluorescence intensity of Cy3 DDPNPs was investigated by adding the gold colloidal nanoparticles with sizes of 20 and 100 nm. Both the gold solutions have an optical density of 1. The quantities of the gold solutions added to 1.5 ml FluoSpheres bead solution are from 1 to 150 μl. The GNPs and Cy3 GNP solutions were characterized using transmission electron microscope (TEM, JEM 1011), fluorescence (Cary Eclipse, Varian) and UV-VIS (JASCO-V570-UV-VIS) spectrophotometers.
3. Results and discussion
Figure 1 presents the transmission electron microscopy (TEM) image and spectra of Cy3 DDPNPs. Beads with size of 100 nm are mono-dispersed in water. The absorption and luminescence spectra peaks are located at 540 and 560 nm, respectively. The absorption spectra of the GNPs are shown in figure 2 with plasmonic resonance absorption peaks at 529 and 563 nm corresponding to GNPs with sizes of 20 and 100 nm, respectively. We expect that the GNPs with absorption peak nearest the absorption peak of Cy3 DDPNPs can enhance the photoluminescence of Cy3 DDPNPs.
Figure 1 TEM image and spectra of Cy3 DDPNPs.
Figure 2 TEM image and absorption spectra of gold colloidal nanoparticles.
The photoluminescence spectra obtained from Cy3 DDPNP solutions containing different amounts of GNPs with size of 20 nm are shown in figure 3(a). We added 0–150 μl GNP solution (4.5×1010 particles per ml) into 1.5 ml Cy3 DDPNP solutions and observed a significant enhancement of fluorescence emission in the Cy3 DDPNPs when GNPs are added into the DDPNP solution. Both spectra exhibit a fluorescence peak at around 560 nm. The spectra shape is mostly unchanged in the presence of the GNPs. The fluorescence emission is enhanced by a factor of 1.5 to 102 with GNPs. The enhancement factor is determined by the ratio of fluorescence intensity of the Cy3 DDPNPs in the presence and absence of GNPs. There is optimal enhancement of the fluorescence intensity of the Cy3 DDPNPs when 70 μl GNP solution was added (figure 3(b)). The photoluminescence intensity of the Cy3 DDPNPs increases when the concentration of GNPs increases (see figure 4).
Figure 3 The luminescence spectra of Cy3 DDPNPs in the presence (a) and absence (b) of 20 nm Au NPs with the quantity of GNP solution changing from 0 to 150 μl in 1.5 ml DDPNP solution under 532 nm excitation.
Figure 4 Fluorescence intensity of Cy3 DDPNPs versus the concentration of 20 nm GNPs.
The 20 nm GNPs cause the luminescence of the Cy3 DDPNPs to increase significantly, whereas the 100 nm GNPs cause less fluorescence enhancement. Figure 5 presents the photoluminescence spectra of the Cy3 DDPNPs in the absence and presence of 100 nm GNPs. The 100 nm GNP concentration dependence of the fluorescence intensity of Cy3 DDPNPs is presented in figure 6. The strongest luminescence intensity of the Cy3 DDPNPs is reached at a GNP concentration of 6.4×107 particles per ml with a fluorophores concentration of 3.64×1010 particles per ml. With the higher concentration of GNPs, the fluorescence intensity becomes saturated and there is no more increase of fluorescence enhancement.
Figure 5 The luminescence spectra of Cy3 DDPNPs in the presence and absence of 100 nm Au NPs with the quantity of GNP solution changing from 0 to 150 μl in 1.5 ml DDPNP solution under 532 nm excitation.
Figure 6 Fluorescence intensity of Cy3 DDPNPs versus the concentration of 100 nm GNPs.
The fluorescence enhancement of the Cy3 DDPNPs caused by gold colloidal particles can be explained by the energy transfer from GNPs to Cy3 DDPNPs. This effect is caused by the interaction between the dipole moments of the fluorophores and the surface plasmon field of the metal which can increase the incident light field, and result in enhanced local fluorescence intensity [1, 4, 5]. Fluorescence resonance energy transfer (FRET) is still known as resonant near field energy transfer between a pair of fluorophores. In the case of the interaction between one fluorophore and one metal, the metal can be used as FRET donors or acceptors corresponding to enhancement or quenching of fluorescence, respectively. In our experiment, the mean distance between GNPs and Cy3 DDPNP is estimated to be about a micrometer in the solution. This distance is too large to have FRET. But the results show that there is a certain interaction that means a GNP can be closer to a Cy3 DDPNP where the far-field interaction between the dipole moments of the fluorophore and the surface plasmon field of the gold occurs. The very strong fluorescence enhancement of the Cy3 DDPNPs caused by 20 nm GNPs is due to the coincidence of surface plasmon resonance wavelength with the fluorophore absorption band. As shown above, the plasmon resonance absorption wavelength of 20 nm gold particles is at 529 nm, while the absorption peak of Cy3 DDPNPs is at 540 nm. This can facilitate the resonance energy transfer from gold particles to Cy3 DDPNPs. The scattering energy from gold can transfer to Cy3 DDPNP so that the excitation on Cy3 DDPNP increases (including direct excitation and indirect excitation from gold particles). It also indicates that the GNPs absorb excitation energy and dipole oscillations are created on the surface of gold metal. The radiating plasmons then appear and excite the fluorophore so that the fluorescence can be enhanced.
In other experimental works larger metal particles can increase the fluorescence enhancement while small metal particles cause less fluorescence enhancement or the quenching of fluorophores. This is because the absorption process is dominant over scattering on small metal particles, but the scattering is dominant on large particles. In our experiments, the 100 nm GNPs cause less fluorescence enhancement of Cy3 DDPNPs than the 20 nm GNPs do. This can be explained as follows: the plasmon resonance absorption wavelength of 100 nm gold particles is at 563 nm, while the absorption peak of Cy3 DDPNPs is at 540 nm and the surface plasmon resonance wavelength coincides less with the fluorophore absorption band. Therefore, the resonance energy transfer ability from gold particles to Cy3 DDPNPs is small. Moreover, the 560 nm emission peak of Cy3 DDPNPs nearly coincides with the plasmon resonance absorption wavelength of 100 nm gold particles (563 nm) so that there can be FRET from the fluorophores to GNPs. This causes the fluorescence enhancement to be small.
4. Conclusion
We have presented our novel results about the photoluminescence enhancement of 100 nm Cy3 DDPNPs by energy transfer from surface plasmons of gold colloidal nanoparticles with sizes of 20 and 100 nm. This is the effect of the resonance energy transfer from GNPs to the fluorophore beads. The interaction between the fluorophores and GNPs is attributed to far-field interaction. The optimal luminescence enhancement of the fluorophores caused by GNPs can be explained as a consequence of the coincidence of the surface plasmon absorption wavelength of GNPs and the absorption wavelength of the Cy3 DDPNPs.
Acknowledgment
This work was supported by the National Foundations for Science and Technology Development (NAFOSTED) No. 103.06.101.09.