Facile Preparation of Au–Ag Composite Nanostructure for High-Sensitive and Uniform Surface-Enhanced Raman Spectroscopy

: Preparation of a high-sensitive and uniform surface-enhanced Raman spectroscopy ( SERS ) active substrate structure usually faces complex processes and high costs. Here, porous Au–Ag composite nanostructures that are conventional fabricated by the deposition of a multilayer Au–Ag, annealing, and dealloying process are proposed for high-performance SERS . By annealing at a suitable temperature, nanopores could be ﬁrmly distributed on the surface, which serves as hot spots. The electric ﬁeld distribution was also performed by the ﬁnite difference time domain. The experiment results exhibited excellent uniformity and high sensitivity of SERS detection. The enhancement factor of the R6G molecules detected by the SERS substrate reached 1.37 × 10 7 , and the relative standard deviation was as low as 4.9%. The minimum detection concentration of R6G molecules by the Au–Ag composite nanostructures with bottom Au mirror could reach 10 − 13 M. The proposed Au–Ag composite nanostructures and the fabrication process have great potential in preparation of a high-sensitive and uniform SERS substrate.


Introduction
Surface-enhanced Raman scattering (SERS) is a nondestructive and ultra-sensitive spectroscopic technique that has been widely used in biomedicine [1][2][3][4], food safety [5][6][7][8][9], environmental monitoring [10,11], etc. An ideal SERS substrate requires excellent signal enhancement, hot spot uniformity, high stability, and facile fabrication [12,13]. SERS enhancement is mainly attributed to electromagnetic (EM) enhancement proportional to the fourth power of the local electric field intensity [14]. Localized surface plasmon resonance (LSPR) generated by metal nanostructures [15][16][17] or deep gaps [18][19][20] demonstrates an extremely strong electric field [21,22], which is widely used in Raman signal enhancement. Nanoparticles, such as nanospheres [23,24], nanotriangles [25], nanorods [26], and nanostars [27,28], could be prepared by chemical methods. However, the hot spots induced by the chemically prepared nanoparticles are randomly distributed due to the random aggregation, resulting in poor uniformity. In contrast, a uniform distribution of hot spots could be obtained through a physical method with ordered and periodic nanostructures [29,30], such as nano-peak arrays, double nano-ring arrays, nano-gratings, and ordered or periodic nanostructures formed by self-assembly. However, preparation of these nanostructures usually requires high manufacturing accuracy equipment, such as electron beam lithography, focused ion beam lithography, and nanoimprint lithography, thus significantly increasing manufacturing costs [31,32]. Furthermore, these methods are always accompanied by limited fabrication areas or long fabrication times. Therefore, it is important to find a facile and large-area preparation method.
The surface structure and the material of the SERS substrate are two key factors affecting Raman signal intensity. For the surface structure, noble metal nanohole structures have attracted extensive attention due to their high density and embedded hotspots. For instance, Koya et al. showed that EM hotspots formed by metal nanoporous materials are widely used in surface-enhanced Raman spectroscopy [33]. Liu et al. fabricated porous gold nanoparticle structures through an ultra-thin alumina membrane incorporated with annealing and dealloying techniques, which showed marvelous uniformity and sensitivity in SERS analysis, and the Raman enhancement factor (EF) reached 1.4 × 10 7 with a relative standard deviation (RSD) less than 6.6% [34]. RSD represents the ratio of standard deviation to average. For the material of the SERS substrate, compared with Au nanoparticles, Ag nanoparticles have stronger local field enhancement factors in the visible light range; however, they have a relatively poor stability. The use of Au-Ag alloy materials can combine the strong plasmonic enhancement of Ag with the material stability of Au. Mandal et al. studied SERS results, showing that the intensity of the SERS substrate with Au-Ag bimetallic nanoparticles is stronger than that with monometallic nanoparticles [35]. Gao et al. reported that the SERS substrate with Au-Ag core-shell nanospheres exhibited strong coupling at a wavelength of 633 nm by a combination of the plasmonic properties induced by the Ag and the chemical stability induced by the Au [36]. However, the preparation process of Au-Ag alloy nanostructures is normally complex and requires nano-processing technology [37][38][39].
In this paper, a facile method is presented that depends on conventional metal deposition annealing and dealloying. Au-Ag nanostructures are formed by alternating the deposition of multilayer gold and silver, annealing at a low temperature, and the dealloying of the as-grown Au-Ag multilayer. By controlling the annealing temperature, the EF of the SERS substrate reached 2.4 × 10 5 . In addition, by adding a gold bottom mirror layer, the sensitivity and electric field strength was further improved, the EF was as high as 1.37 × 10 7 , and the RSD reached 4.9%.

Sample Fabrication
The fabrication process of the composite Au-Ag alloy structure is shown in Figure 1. For sample 1, the preparation process was as follow: a 0.5 nm-thick Ti was first evaporated onto the Si substrate at a rate of 0.5 Å s −1 . Then, four layers of 10 nm Au and 20 nm Ag were alternately deposited; the surface Au-Ag alloy was formed by annealing for 30 s under nitrogen flow. The annealed porous Au-Ag alloy structure was immersed in the H 3 PO 4 etching solution for 1 min to remove the Ag at room temperature, and then immediately washed by DI water. Finally, a large-scale surface porous Au-Ag alloy substrate was successfully finished.

Structural Characterization and SERS Measurements
The surface morphology of the samples was observed by a field emission scanning electron microscope (SEM), and the proportion of Au and Ag components on the surface of the samples was analyzed by an X-ray energy spectrometer. A rhodamine 6G (R6G) molecule was introduced as a probe molecule to characterize the SERS substrate perfor- Here, the Au-Ag multilayer was adopted to fuse the metals more fully after annealing, resulting in denser and uniform holes after dealloying Ag. The preparation process of sample 2 is shown in the Figure 1b. Compared with sample 1, an additional bottom Au layer and an SiO 2 separation layer were added beneath the porous Au-Ag alloy. Then, the 60 nm bottom Au, 30 nm SiO 2 , and 0.5 nm Ti layers were deposited. Finally, a porous Au-Ag alloy substrate with bottom Au was successfully prepared after the annealing and dealloying of the top Au-Ag multilayer.

Structural Characterization and SERS Measurements
The surface morphology of the samples was observed by a field emission scanning electron microscope (SEM), and the proportion of Au and Ag components on the surface of the samples was analyzed by an X-ray energy spectrometer. A rhodamine 6G (R6G) molecule was introduced as a probe molecule to characterize the SERS substrate performance of the Au-Ag composite nanostructures. The R6G aqueous solutions with different concentrations (10 −6~1 0 −13 M) were prepared. The samples were immersed in the prepared aqueous solution for 2 h and then dried by nitrogen flow. The Raman signals of the two samples were characterized by a 532 nm laser confocal Raman microscope with a laser power of 10 mW. The signal acquisition time was 9 s for R6G and the acquisition process was cycled three times to remove the spike noise.

Simulations
Three-dimensional finite-difference-time-domain (FDTD) was used to simulate the near-field EM field distribution. All boundaries were equipped with a perfectly matched layer (PML) condition. A plane wave with a wavelength of 532 nm as used to illuminate from the top, and the incident wave propagated perpendicular to the direction of the base surface. Field strength monitors were placed on the horizontal and vertical sections of the substrate to obtain the field strength distribution. The dielectric constants (ε) of Au-Ag alloys are represented by ε(α) = αε Au + (1 − α) ε Ag , where α = 0.385 (corresponding to the Au content in the Au-Ag alloys), and the dielectric constants of Au and Ag are taken from the data of Palik [40].

Results and Discussion
Figure 2a-e shows the SEM images of the Au-Ag composite nanostructures in sample 2 after annealing and dealloying processes. Sample 1 and sample 2 have a similar surface morphology at the same annealing temperature. From the intuitive view of the SEM images, Figure 2b shows that there is a thin layer of nano-network structure on the surface and Figure 2c shows a pore structure. The surface morphology of substrates under 200 • C and 300 • C was much more uniform than that of substrates at other temperatures. At the low annealing temperature, the porous structure could not be formed on the surface of the dealloyed structure and the Au and Ag were immersed in the etching solution. When the annealing temperature was greater than 400 • C, the Au-Ag structure after dealloying treatment formed a large area, and Ag in the alloy was difficult to be corroded by the etching solution to form a porous structure. Figure 2f shows the qualitative and quantitative analysis of the surface composition of the sample after annealing and dealloying by an X-ray energy dispersive spectrometer. It can be seen that as the annealing temperature increased, the proportion of Ag in the alloy increased, again confirming that the higher the annealing temperature, the more difficult it is to corrode the Ag in the alloy. A layer of Ti was needed as the adhesion layer between the Au-Ag structure and the Si substrate; otherwise, the structure may be unstable in the dealloying stage.
The SERS performance of the Au-Ag composite structure at different annealing temperatures is shown in Figure 3a. It was found that the SERS intensity first increased and then decreased with the increase in the annealing temperature. The intensity was sharply decreased with an annealing temperature greater than 300 • C. In addition, we digitally marked important bands in the Raman spectra. As shown in Figure 3b, the Raman intensity The Raman intensities of these peaks had a consistent increasing trend and the best SERS signal was obtained when the temperature was at 300 • C. of Ti was needed as the adhesion layer between the Au-Ag structure and the Si substrate; otherwise, the structure may be unstable in the dealloying stage. The SERS performance of the Au-Ag composite structure at different annealing temperatures is shown in Figure 3a. It was found that the SERS intensity first increased and then decreased with the increase in the annealing temperature. The intensity was sharply decreased with an annealing temperature greater than 300 °C. In addition, we digitally marked important bands in the Raman spectra. As shown in Figure 3b, the Raman intensity peaks at the 603, 765, and 1178 cm −1 are plotted as a function of the annealing temperature. The Raman intensities of these peaks had a consistent increasing trend and the best SERS signal was obtained when the temperature was at 300 °C.  otherwise, the structure may be unstable in the dealloying stage. The SERS performance of the Au-Ag composite structure at different annealing temperatures is shown in Figure 3a. It was found that the SERS intensity first increased and then decreased with the increase in the annealing temperature. The intensity was sharply decreased with an annealing temperature greater than 300 °C. In addition, we digitally marked important bands in the Raman spectra. As shown in Figure 3b, the Raman intensity peaks at the 603, 765, and 1178 cm −1 are plotted as a function of the annealing temperature. The Raman intensities of these peaks had a consistent increasing trend and the best SERS signal was obtained when the temperature was at 300 °C.  Here, the FDTD simulation method was also used to analyze the distribution and intensity of the enhanced EM field on the surface of the SERS substrate. Figure 4a shows a simulation of the substrate without annealing and etching and Figure 4b,c shows an electric field (Re (|E|) distribution cross-section (X-Y plane) of the composite nanostructures and the composite nanostructures with a bottom Au reflector, respectively. Compared with Figure 4b,c, it is obvious that the Au-Ag alloy nanostructures of sample 1 and sample 2 show a huge local field enhancement effect. The local field enhancement shown in Figure 4c was stronger than that shown in Figure 4b, which was due to the bottom Au layer reflecting Photonics 2023, 10, 354 5 of 8 the light passing through the porous surface, enhancing the absorption [41]. Thus, the local field enhancement of the porous surface and the collection efficiency of SERS were improved. From the FDTD simulation, we demonstrated that the electric field intensity of Au-Ag nanostructures with gold mirrors was, on average, about 3.3 times that of the Au-Ag nanostructures without the bottom mirror.
intensity of the enhanced EM field on the surface of the SERS substrate. Figure 4a a simulation of the substrate without annealing and etching and Figure 4b,c sh electric field (Re (|E|) distribution cross-section (X-Y plane) of the composite nan tures and the composite nanostructures with a bottom Au reflector, respectively pared with Figure 4b,c, it is obvious that the Au-Ag alloy nanostructures of samp sample 2 show a huge local field enhancement effect. The local field enhancement in Figure 4c was stronger than that shown in Figure 4b, which was due to the bott layer reflecting the light passing through the porous surface, enhancing the abs [41]. Thus, the local field enhancement of the porous surface and the collection effi of SERS were improved. From the FDTD simulation, we demonstrated that the field intensity of Au-Ag nanostructures with gold mirrors was, on average, about 3 that of the Au-Ag nanostructures without the bottom mirror. In order to verify the effect of the Au-Ag composite nanostructure on the signal enhancement, the substrates were immersed in R6G aqueous solutions of d concentrations and dried with nitrogen after 7 h. The Raman signal of the samp characterized by a confocal Raman microscope with a 532 nm laser, and the inte time was 5 s. It can be seen from Figure 5a,b that the Raman signal intensity de with the dilution of the concentration; Raman signals can also be observed for bo ples as the concentration was as low as 10 −11 M. This indicates that the composite structure is very sensitive to R6G molecules. The minimum detectable concentr the composite Au-Ag nanostructures with the bottom Au mirror can reach 10 −13 M characteristic Raman peaks of R6G at 603, 765, and 1178cm −1 were selected to bu calibration curves of the Raman intensities as a function of R6G concentrations. The show that the Raman intensity of the three characteristic peaks has a good linear tion with the logarithmic concentration, and the correlation coefficients wee 0.985 and 0.950, respectively. which proved that the Au-Ag composite nanostructure w tom Au layer can obtain an ultra-low detectable solubility of R6G molecules. The d of ultra-low concentration analytes shows that the high-sensitivity SERS could be r by the porous Au-Ag composite nanostructure. In addition, compared with the interference lithography [21] or 3D lithography [29], the preparation process used paper does not need to use sophisticated optical instruments. In order to verify the effect of the Au-Ag composite nanostructure on the Raman signal enhancement, the substrates were immersed in R6G aqueous solutions of different concentrations and dried with nitrogen after 7 h. The Raman signal of the samples was characterized by a confocal Raman microscope with a 532 nm laser, and the integration time was 5 s. It can be seen from Figure 5a,b that the Raman signal intensity decreased with the dilution of the concentration; Raman signals can also be observed for both samples as the concentration was as low as 10 −11 M. This indicates that the composite Au-Ag structure is very sensitive to R6G molecules. The minimum detectable concentration of the composite Au-Ag nanostructures with the bottom Au mirror can reach 10 −13 M; three characteristic Raman peaks of R6G at 603, 765, and 1178cm −1 were selected to build the calibration curves of the Raman intensities as a function of R6G concentrations. The results show that the Raman intensity of the three characteristic peaks has a good linear correlation with the logarithmic concentration, and the correlation coefficients wee 0.985, 0.979, and 0.950, respectively. which proved that the Au-Ag composite nanostructure with bottom Au layer can obtain an ultra-low detectable solubility of R6G molecules. The detection of ultra-low concentration analytes shows that the high-sensitivity SERS could be realized by the porous Au-Ag composite nanostructure. In addition, compared with the use of interference lithography [21] or 3D lithography [29], the preparation process used in this paper does not need to use sophisticated optical instruments. Here, the SERS and non-SERS spectra of the R6G molecules were measured, and EF of the Au-Ag composite nanostructure were estimated by comparing the Raman tensity of the SERS signal and the non-SERS signal, as follows. Here, the SERS and non-SERS spectra of the R6G molecules were measured, and the EF of the Au-Ag composite nanostructure were estimated by comparing the Raman intensity of the SERS signal and the non-SERS signal, as follows.
where C SERS represents the concentration of R6G molecules immersed in aqueous solution and I SERS represents the Raman intensities of the R6G molecule adhered to the Au-Ag composite nanostructure. Here, we measured the Raman spectrum of a silicon wafer treated by the R6G solution (0.5 × 10 −5 M) and the SERS spectrum of the Au-Ag composite nanostructure sample treated by the R6G solution (10 −12 M); at 765 cm −1 , I SERS , I non were 1462 and 534, respectively. Therefore, the EF of the 765 cm −1 Raman peak was calculated to be about 1.37 × 10 7 for the composite Au-Ag nanostructures. The EF of the Au-Ag composite nanostructure was much larger than that in many earlier reports with respect to porous Au-Ag hybrid nanoplates (EF = 1.4 × 10 6 ) [22] and nano-sponges (EF = 6.4 × 10 5 ) [42]. In addition, the EF of the Raman peak at 765 cm −1 for the Au-Ag nanostructures without underlying gold mirrors was estimated to be 2.4 × 10 5 . This indicated that high-sensitivity SERS substates could be achieved by the facile fabrication of the Au-Ag composite nanostructures. In addition, the uniformity is also an important parameter in special applications for the SERS substrate. 10 −6 M R6G was taken as the molecule of detection. Figure 6a shows that the scanning area of a random position with an area of 50 × 50 µm 2 on the substrate was performed to estimate the uniformity, and 100 detection points was uniformly distributed on this square area. The Raman map of R6G at 765 cm −1 is shown in the Figure 6b, illustrating significant signal uniformity of the SERS substrate with the Au-Ag composite structure. Additionally, the average RSD of Raman intensity for the 765 cm −1 is 4.9%. The results indicate that a uniform SERS could be formed by the porous Au-Ag composite nanostructures. Here, the SERS and non-SERS spectra of the R6G molecules were measured, and the EF of the Au-Ag composite nanostructure were estimated by comparing the Raman intensity of the SERS signal and the non-SERS signal, as follows. (1) where CSERS represents the concentration of R6G molecules immersed in aqueous solution and ISERS represents the Raman intensities of the R6G molecule adhered to the Au-Ag composite nanostructure. Here, we measured the Raman spectrum of a silicon wafer treated by the R6G solution (0.5 × 10 −5 M) and the SERS spectrum of the Au-Ag composite nanostructure sample treated by the R6G solution (10 −12 M); at 765cm −1 , ISERS, Inon were 1462 and 534, respectively. Therefore, the EF of the 765 cm −1 Raman peak was calculated to be about 1.37 × 10 7 for the composite Au-Ag nanostructures. The EF of the Au-Ag composite nanostructure was much larger than that in many earlier reports with respect to porous Au-Ag hybrid nanoplates (EF = 1.4 × 10 6 ) [22] and nano-sponges (EF = 6.4 × 10 5 ) [42]. In addition, the EF of the Raman peak at 765 cm −1 for the Au-Ag nanostructures without underlying gold mirrors was estimated to be 2.4 × 10 5 . This indicated that high-sensitivity SERS substates could be achieved by the facile fabrication of the Au-Ag composite nanostructures.
In addition, the uniformity is also an important parameter in special applications for the SERS substrate. 10 −6 M R6G was taken as the molecule of detection. Figure 6a shows that the scanning area of a random position with an area of 50 × 50 µm 2 on the substrate was performed to estimate the uniformity, and 100 detection points was uniformly distributed on this square area. The Raman map of R6G at 765 cm −1 is shown in the Figure  6b, illustrating significant signal uniformity of the SERS substrate with the Au-Ag composite structure. Additionally, the average RSD of Raman intensity for the 765 cm −1 is 4.9%.
The results indicate that a uniform SERS could be formed by the porous Au-Ag composite nanostructures.

Conclusions
In summary, this work developed a facile fabrication process route for a high-performance SERS substrate. After the deposition of the multilayer Au-Ag structure, annealing and dealloying techniques were performed to form a porous Au-Ag composite nanostructure. The results showed that the local electric field can be further enhanced by the SERS effect and the reflection of the underlying bottom Au. Furthermore, the corresponding results were also confirmed in the experiment. Compared with the use of modern precision optical instruments, the structure used in this paper can be prepared in a large area and the preparation process cost is lower. The EF of the R6G molecule detected by the SERS