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Nanoporous gold (NPG) has been reported to provide remarkable fluorescence enhancement of adjacent fluorophores due to the metal-enhanced fluorescence phenomenon (MEF), and the enhancement is related with the characteristic length of nanoporosity. To fully understand the effect of NPG on nearby fluorophores, it is desirable to study systems with well-defined metal-fluorophore distances. In this study we investigated the distance effect by using silica as the spacing layer between fluorophores and NPG. Originating from competition between plasmonic amplifying and metallic quenching, the dye molecule rhodamine 6G was best enhanced by 20-nm SiO2 coated nanoporous gold with the pore size of 36 nm, while the protein phycoerythrin was best enhanced by 15-nm SiO2 coated nanoporous gold with the pore size of 42 nm and the quantum dots were best enhanced by 20-nm SiO2 coated nanoporous gold with the pore size of 42 nm.
© 2017 Optical Society of America
1. Introduction
Nanostructured noble metals exhibit remarkable optical properties due to the excitation of localized plasmon, which have been widely utilized to control a variety of optical signals [1–11]. The capability to enhance the fluorescence intensities of the nearby fluorescent species has stimulated considerable interest due to the promising applications of plasmonic based trace molecular detection, as well as biological sensing and imaging, and extensive researches have been carried out on the fluorescence enhancement with nanostructured materials. Several pioneering works demonstrated that the enhancement of the fluorescence is related with chemical nature, size and shape of the nanostructure, the coincidence between the nanostructure resonance wavelength and the fluorophore adsorption/emission wavelength, and the distance separating the fluorophore and metal surface [12,13]. The size and the shape decide the local electromagnetic field surrounding the metallic nanostructure which allows concentration of optical field for enhanced fluorescence, while the distance between fluorophore and metal surface decides competition of the enhancement and quenching of the fluorescence. For short distance, quenching of emission is well-known and is attributed to damping of dipole oscillators and coupling of energy into evanescent waves at the surface of the metal, while for distanced far, fluorescence is not affected and behaves as in free space [14–19]. Thus, a certain distance from the plasmonic surface is preferred, where energy transfer into plasmon is reduced but the electromaganetic field strength can still be great enough to enhance fluorescence emission [20,21]. To enhance the fluorescence and eliminate the quenching, diverse methods have been developed to fabricate metallic nanostructures with high electric fields and an insulator medium separator between fluorophores and metal surface, and in the distance 10-20 nm away from the plasmonic surface, strong enhancement has been observed [20–23].
Compared with the other available metal enhanced fluorescence substrates, nanoporous gold (NPG) possess intriguing properties that offer potential benefits for applications, such as high specific surface area, continuously tunable quasi-periodic ligament and nanoporous channels [24], excellent chemical stability and et al. [25–33]. Dramatically enhanced intensities of single molecule has been observed on as-prepared NPG film [29], and with 3-5 nm protein coated, the enhancement of ensemble dye molecule from as-dealloyed NPG can be enhanced to ~50-fold [30]. According to the optimal distance between the plasmonic surface and fluorophore reported before, better enhancement is promising if thicker separation layer located on the surface of NPG. Silica, one of the extensively studied biomaterial with a surface can be chemically modified with other ligands, is an excellent candidate for such coating.
In this study, NPG films were covered by a thin dielectric silica layer. Unlike protein or polyelectrolyte surroundings, silica was much more stable and easily controlled. Since the fluorophore emission on the metal surface is a competition of the surface plasmon enhancement and energy transfer quenching [34–36], silica thin layer with selective thicknesses were fabricated on NPG films that with different ligament and nanopore sizes. Moreover, to gain more insight into the phenomenon and to fully understand the enhancement rule of NPG, three different kinds of fluorophore were applied. Strong near-field excitation induced by NPG and minimized quenching effect by silica internal layer enabled around 70 fold and 33 fold enhancement of dye molecule and fluorescent protein, and the fluorescence from QDs was also obviously enhanced by 23 times.
2. Experimental
NPG films with a thickness of ~100 nm were fabricated by dealloying Ag65Au35 (atomic ratio) leaves (purchased from Sepp Leaf Products. Inc). With selectively etching silver component from the alloy by 68% nitric acid, an open bicontinuous three dimensional nanoporous hierarchical structure expands to the entire film and the ligament and pore size increases with extending of the etching time (Fig. 1) [23–28]. After carefully rinsed with deionized water, the as-prepared NPG films were transferred onto the polymer sheet surface and dried in the air for further experiment.
Fig. 1 Top-view SEM micrographs of dealloyed NPG films with an average pore size of (a) ~22nm,(b) ~33nm,(c) ~45 nm. (d) Tunable nanopore size as a function of etching time. The larger pore size resulted from longer etching time and the largest pore size is ~50 nm due to the thickness of Ag65Au35 alloy film is 100 nm.
SiO2 films were deposited on the surface of the NPG films by physical vapour deposition (optorun OTFC900) at 200°C under pressure of 7 × 10−4 Pa with evaporation rate of 0.3 nm/s. The thickness of SiO2 was controlled by depositing time.
Microstructure characterization and material property analysis were accomplished by using a scanning electron microscope (SEM, FEI Versa3D), a transmission electron microscope (TEM, JEM-ARM200F), and a UV-Vis spectrophotometer (HORIBA Dual-FL-UV-800). Fluorescence was detected by a spectrometer with 532-nm laser excitation and the laser power at the sample surface is about 1 mW.
Rhodamine 6G (R6G) and phycoerythrin (R-PE) were purchased from Aladdin, and quantum dots was purchased from China Beijing Beibang Science & Technology Co. Ltd. A 10−6 M R6G aqueous solution, 0.04 mg/ml R-PE aqueous solution and 50 nM quantum dots aqueous solution were used in the experiment. The polymer, bare NPG films and SiO2 coated NPG (SiO2@NPG) films were immersed in fluorophore solution for several hours in order to stablize the fluorescent molecules on the substrate, and the unbounded molecules were carefully removed by washing with the deionized water. After drying in the air, the samples were measured by the fluorescence spectrometer.
3. Results and discussions
Since the electromagnetic field intensity varies with the scale of the porous structure [27–35], NPG films with tunable nanoporosity were fabricated as precursors, and the representive SEM images of NPG with selective ligament size of 22 nm (NPG22), 33 nm (NPG33) and 45 nm (NPG45) are shown in Fig. 1(a)-1(c), respectively. The evolution of the NPG ligaments and nanopore sizes with etching time is given in Fig. 1(d), and longer dealloying time leads to larger nanopore size due to the self-assemble of the Au atoms during etching process. The ligament and nanopore sizes are calculated by processing a Fourier-transformation of the selected region from the SEM images (see Fig. 7 in the Appendix) [25].
To eliminate the quenching effect due to the energy transfer between the gold and fluorophore, silica films with various thicknesses (5, 10, 15, 20 and 25 nm, respectively) were deposited on the surface of the NPG films as the space layer. As shown in Fig. 2(a), SiO2 film uniformly covered on the gold ligaments, and the thickness is coincident with the numerical value that we designed (see Fig. 2(b)).
Fig. 2 (a) SEM images of 10 nm-SiO2 coated NPG film, and the measured thickness is coincident with the manufactured value by physical vapour deposition. (b) TEM micrograph of selected SiO2@NPG film with the nanopore and ligament sizes of ~42 nm and SiO2 coating of 10 nm. (c) The absorption spectra of bare NPG films with different nanopore sizes. (d)The absorption spectra of 15 nm-SiO2 coated NPG films with different ligament and nanopore sizes.
Typical UV-Vis absorption spectra of the bare NPG films and SiO2@NPG films are presented in Fig. 2(c) and 2(d), respectively. Regardless of the nanopore sizes and the thickness of silica films, two characteristic peaks are shown in each spectrum. The lower wavelength peaks located at ~495 nm, originated from the resonant absorption of gold films, do not change with the pore size [36], and it is also independent of the silica coating. In contrast, the higher wavelength peaks, arisen from the local SPR, represents the significant redshift from ~538 nm to ~583 nm with the increasing of pore sizes of bare NPG from ~22 nm to ~46 nm, and more redshift occurs with silica coating due to the increase of the refractive indices of the SiO2 media.
To evaluate the fluorescence enhancement of the SiO2@NPG films with tunable nanoporosity and silica coating, Rhodamine 6G (R6G) fluorophore with the absorption and emission peaks at ~524 nm and ~553 nm was chosen and stabilized on polymer substrate, SiO2 coated polymer, NPG films and SiO2@NPG films. Figure 3(a) shows the fluorescence spectra of R6G on silica coated NPG36 (SiO2@NPG36), and the spectra of R6G on bare NPG and on polymer substrate are also shown for compare. The fluorescence intensities of R6G on SiO2@NPG are consistently higher than those on the bare NPG and polymer, and the fluorescence intensity enlarged with the distance changing from 0 to 20 nm and then dropped at the distance of 25 nm, getting the biggest enhancement when the distance is 20 nm. The enhancement factor was defined and calculated by dividing fluorescence intensity on SiO2@NPG with that on control sample, where polymer was chosen as controller (more details can be found in the Appendix Fig. 8). The enhancement factor of NPG36 and SiO2@NPG36 are showed in the insert of Fig. 3(a), and the maximum enhancement factor of R6G on NPG36 is as high as ~70, which is obtained on 20-nm silica coating.
Fig. 3 Fluorescence enhancement of R6G on polymer and NPG films with various silica coating, 0, 5, 10, 15, 20, and 25 nm, respectively. Inset shows the fluorescence enhancement factor, which is determined by the ratio between the peak intensity of R6G at 553 nm on SiO2 coated porous films (SiO2@NPG36) and that on polymer. The largest enhancement factor is about 70 from the 20 nm silica coated NPG36 film. (b) Histogram of the fluorescence intensities from R6G on polymer, bare NPG films and SiO2@NPG films. The fluorescence intensities which are determined by the height of R6G emission peak at ~553 nm, and different coloured rectangles are used to distinguish NPG films with different pore sizes.
It can be seen that, the fluorescence intensity from the polymer was very weak, and the porous structure obviously enhanced the fluorescence intensity about 2 times. With silica coating the fluorescence intensity can be further enhanced, and the enhancement factor depends both on the characteristic length of the NPG and also the thickness of the silica space layer. Moreover, the variation of the fluorescence intensities from R6G on NPG with different pore sizes and silica coating were measured to further check the size effect, and the results are shown in Fig. 3(b). Obviously, for the NPG with same ligament size, a significant and continuous increase of fluorescence intensity was observed with the increasing of silica thickness from 5 nm to 20 nm and then decrease after. The change trend independent on the nanopore size of NPG, suggesting the optimum distance for R6G fluorescence enhancement with NPG is around 20 nm.
To confirm the change trend of the fluorescence enhancement from SiO2@NPG films, other two fluorophores, phycoerythrin (R-PE) and CdTe quantum dots(QDs), were used to repeat the fluorescence measurement. Both the absorption and emission wavelengthes of R-PE and QDs overlap with the surface plasmon position (SPP) of SiO2@NPG (see Fig. 4).
Fig. 4 Absorption spectrum of a bare NPG36 and NPG36 covered with 20-nm-thick SiO2 film, and the absorption and emission peaks position of R6G, R-PE and QDs (only Emission). (a) Green, Dark Cyan bars indicate the absorption/emission for R6G molecule and Black bar indicates the excitation in the experiment. (b) Olive, Cyan bars indicate the absorption/emission for R-PE protein and Black bar indicates the excitation in the experiment. (c) Blue bar indicates the emission for QDs and the Black bar indicates the excitation in the experiment. As the absorption of QDs is a decline curve, the excitation (532nm) can motivate the QDs well.
R-PE were deposited by exposing the films to 0.04 mg/ml R-PE aqueous solution for 12 hours [37], and QDs were stabilized on the films by immersing in 50 nM QDs aqueous solution for 12 hours (SEM energy disperse spectroscopy was used to verify the loading amount of QD, and more details can be found in Appendix Table 1). And then all specimens were quickly washed by deionized water to remove the unbound fluorophores and dried in the air for measuring. The corresponding fluorescence results are shown in Fig. 5 and the histograms of the varied fluorescence intensities obtained from the specimen based on NPG with different ligament size are shown in Fig. 6. At the identical testing condition used as those of R6G, the fluorescence intensities of R-PE adsorbed on NPG and SiO2@NPG were obviously enhanced, but exhibited weaker enhancement than that of R6G (see Fig. 5(a) and 5(b)). Moreover, the maximum enhancement was obtained with 15-nm SiO2 coated NPG42, which is different from that for R6G (see Fig. 6(a)).
Table 1. Energy Disperse Spectroscopy (EDS) analysis of QDs decorated NPG and SiO2@NPG films.
Fig. 5 The fluorescence spectra and fluorescence enhancement factor of R-PE and QDs on polymer and NPG films with various silica coating, 0, 5, 10, 15, 20, and 25 nm, respectively. (a) The fluorescence spectra of R-PE on NPG42 films with silica coating. (b) The fluorescence enhancement factor of R-PE, which is determined by the ratio between the peak intensity of R-PE at ~575 nm on SiO2 coated porous films (SiO2@NPG42) and that on polymer. (c) The fluorescence spectra of QDs on NPG42 films with silica coating. (d) The fluorescence enhancement factor, which is determined by the ratio between the peak intensity of QDs at ~630 nm on SiO2 coated porous films (SiO2@NPG42) and that on polymer. The numbers in (a) and (c) are the thicknesses of silica.
Fig. 6 Histogram of the fluorescence intensities from R-PE and QDs on different substrates. (a) Fluorescence intensity of R-PE on polymer, bare NPG and SiO2@NPG films. (b) Fluorescence intensity of QDs on polymer, bare NPG and SiO2@NPG films.
For the QDs, the fluorescence intensities on NPG and SiO2@NPG were also enhanced, but the enhancement was even weaker than that of R-PE. Additionally, the maximum enhancement was obtained with 20-nm SiO2 coated NPG42 (see Fig. 6(b)), which is different from that for R6G and R-PE, either in silica thickness or in characteristic NPG ligament size. The best coating thickness for R6G and QDs fluorescence enhancement is 20nm, which is larger than that for R-PE (15 nm). It is known that R-PE is a fluorescent protein which has a chromophore clad by a layer of protein, and the protein layer also works as spacer which can increase the distance between the chromophore and the substrates. Thus, the best coating thickness of NPG for MEF should around 20 nm, which is similar with silica-coated gold nanorod [21]. However, the optimal ligament size of NPG is related with the size of the fluorophore. From the results it can be seen that the best ligament size for both R-PE and QDs is 42 nm, while that for R6G is 36 nm.
From the investigate of the three kinds of the fluorophores, it can be concluded that: (1) silica coating can efficiently enhance the fluorescence enhancement of NPG films, and the enhancement level is related with the characteristic length of NPG; (2) for fluorophore enhancement, there exists an optimum silica coating thickness, and an optimum ligament size of NPG as well; (3) the best distance for MEF is 20 nm far from the surface of NPG, and the optimum characteristic length of NPG is dependent on the probe molecule, larger fluorophore needs NPG with larger ligament.
4. Conclusion
In summary, we developed a simple but efficient method to prepare plasmonic substrate for fluorescence enhancement. SiO2 coating can significantly improve the enhancement ability of NPG films, and the best spacer thickness for MEF is around 20 nm. Meanwhile, the fluorescence enhancement level of the SiO2@NPG substrates with different pore sizes and various distances between fluorescence probes were systemically studied, and the SiO2@NPG with ligament around 40 nm shows the best enhancement. In general,the experimental observations are important for NPG on fluorescence enhanced sensing. Additionally, these quantitative measurements will help in understanding the fluorescence enhancement mechanism of nanoporous gold, and in modeling and deducing its physical performances in plasmonic applications.
Appendix
The characteristic length scale was calculated with the formula:
Where D is the diameter of the ligament and nanopore, and B is the pixel distance which is the average of the max and min distance between 0 to the highest column.As shown in the Fig. 7, the sample possesses a characteristic length scale around 50 nm that is associated with a statistical period length of the structure and equals the sum of the average ligament diameter and average pore diameter. Thus, the average ligament for both ligament and pore should be 25 nm. Since the diameter of the characteristic varies a little due to the selected region, several regions on NPG film will be chosen for calculation, and the used ligament size in the work is the average value of 5 different regions.
Fig. 7 (a) SEM iamges of nanoporous gold. (b) Fourier-transformed pattern showing the quasiperiodic feature of nanoporous gold. The inserted intensity profile was taken along the dash arrowhead in the Fourier-transform pattern [25].
Fig. 8 (a) Fluoresce spectra of R6G on the polymer substrate with silica coating. (b) Fluoresce spectra of R-PE on the polymer substrate with silica coating.
As shown in the figure, the fluorescence intensity regardless on the silicon coating. Thus, for easy compare, bare polymer was chosen as the control sample for the enhancement factor calculation.
We verified the QD amount through SEM energy disperse spectroscopy (EDS), and found that a similar number of QDs were deposited on the NPG substrates with and without silica coating. For the as-dealloyed NPG, the loading amount of QD decreases slightly with the increasing of the ligament size due to the decreasing of surface to volume ratio; for the silica coated NPG, the loading amount increases with ligament size due to the increasing of the surface area with silica spacer. Thus, the loading capacity of fluorophores on the SiO2@NPG does not reduce due to the silica coating. The possible reason is due to the hydrophobic property of the NPG film (which didn’t discuss in this work). In such case, most of the fluorophores in aqueous solution (which were used in our experiment) only can attach on the surface layer of the ligament, and silicon coating seldom influence the effective surface area.
Funding
National Key Scientific Instrument Project (2012YQ150092); National Natural Science Foundation of China (11434005&61675133); Shanghai Municipal Science and Technology Commission (14JC1401600).
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