*Characterization of Ag@Au NPs*

We studied the morphology and size of Ag@Au nanoparticles via SEM and TEM. Figure 1a is the SEM image of Ag@Au nanoparticles prepared when the amount of silver seed added is 40 µL, and Figure 1b is the corresponding TEM image. As we can see, the surface of the prepared Ag@Au nanoparticles is distributed with dense, thorn-like structures. From the HRTEM image (Figure 1c) of the gold thorns grown on the surface, it can be seen that the lattice spacing is 0.24 nm, indicating that the exposed crystal faces of the gold thorns are mainly composed of {111} planes [4]. Figure 1d shows the corresponding HAADF map. We performed EDS characterization of the prepared Ag@Au nanoparticles, as shown in Figure 1e–g. Figure 1e,f are the distribution diagrams of silver and gold, respectively. Figure 1g shows the overlapping distribution of the two elements. From the EDS results, it can be seen that the atomic ratio of gold to silver is 68:32.

**Figure 1.** (**a**) SEM, (**b**) TEM, (**c**) HRTEM, and (**d**) HAADF images of as-prepared Ag@Au NPs, when the amount of Ag seed was 40 uL. (**e**–**g**) The corresponding EDS images.

Figure 2 shows the characterization results of Ag@Au nanoparticles obtained when the amount of silver seed added is 500 µL. Figure 2a,b show the SEM and TEM images of Ag@Au nanoparticles, respectively. From the figure, we can clearly see that the Ag@Au nanoparticles obtained still maintain a spherical structure, and have a uniform size distribution of about 95 nm. From its HRTEM image (Figure 2c), we can see that the lattice spacing of gold nanoparticles distributed on the surface is 0.24 nm, indicating that it is mainly composed of {111} planes. In addition, from the HAADF map (Figure 2d), it can be seen that only a small part of the Ag@Au nanoparticles are gray in the central area, indicating that the Ag@Au nanoparticles have formed a partially hollow structure, while the main part is still a solid structure. Furthermore, we collected the distribution of Au and Ag elements in Ag@Au nanoparticles via EDX, as shown in Figure 2e–g. From the figure, we can see that gold and silver form an alloy structure distributed over the entire surface.

**Figure 2.** (**a**) SEM, (**b**) TEM, (**c**) HRTEM, and (**d**) HAADF images of as-prepared Ag@Au nanoparticles, when the amount of Ag seed was 500 µL. (**e**–**g**) The corresponding EDS images.

Furthermore, we explored the effects of the amount of silver seeds on the Ag@Au nanoparticles. As shown in Figure 3, when there was no silver seed, we prepared a mesoporous gold structure (Figure 3a). When adding 40 µL of silver seeds, we prepared Ag@Au nanoparticles with dense, thorn-like structures on their surface (Figure 3b). When the amount of silver seeds was doubled (80 µL), the thorn-like structures on the surface of the Ag@Au nanoparticles were reduced (Figure 3c). When the amount of silver seeds was further increased to 160 µL, the thorn-like structures on the surface were further reduced (Figure 3d). When the amount of silver seed was increased to 500 µL, we achieved a spherical structure with shape retention (Figure 3e). According to the EDS characterization results of the nanoparticles prepared under different concentrations of silver seeds, we performed a statistical analysis of the proportions of gold and silver atoms, as shown in Figure 3f. The EDS diagrams of Ag@Au nanoparticles prepared under different silver seed conditions are shown in Figure A1. Analyzing the EDS results, when the amount of silver seeds added was 40, 80, 160, and 500 µL, the proportion of tightness in the prepared Ag@Au nanoparticles was 0.68, 0.44, 0.38, and 0.20, respectively. The corresponding proportions of silver were 0.32, 0.56, 0.62, and 0.80, respectively.

Based on the above experimental results, we can make the following inferences on the formation mechanism of Ag@Au nanoparticles: Figure 3g is a schematic diagram of the growth of Ag@Au nanoparticles. First, Au3+ ions in HAuCl4·3H2O solution combine with Br<sup>−</sup> ions in CTAB to form [AuBr4] <sup>−</sup>, and then ascorbic acid reduces [AuBr4] − to get Au<sup>+</sup> . This process is consistent with previous reports [32,33]. When silver seeds modified with glutathione were added, Au<sup>+</sup> reduced glutathione adsorbed on the surface of silver seeds in situ to form Au<sup>0</sup> . It can be seen from Figure 2D that Au<sup>+</sup> inevitably had a galvanic replacement reaction with silver seeds (forming few hollow structures). However, the majority of Ag@Au nanoparticles are still solid, which also indicates that the glutathione on the surface of the silver seeds largely inhibits the galvanic replacement reaction. In addition, the reduction of glutathione also causes the gold ions to be reduced to gold atoms and deposited on the surface of the silver seeds, which also prevents the occurrence of the galvanic replacement reaction. Thus, when the amount of silver seeds is low, there are enough gold atoms to grow along the direction of glutathione, and eventually form seaurchin-like Ag@Au nanoparticles. When the amount of silver seeds is greater, the Ag@Au spherical shell structure with the original shape will be formed. The above results show that we have successfully prepared Ag@Au nanoparticles. Compared with the previously reported Au@Ag core–shell structure and the Ag@Au hollow structure prepared by the

displacement reaction, the Ag@Au nanoparticles we prepared have a solid structure. A gold thorn structure grows on the surface of the seed, which realizes the combination of the advantages of gold and silver nanoparticles.

**Figure 3.** TEM images of as-prepared Ag@Au nanoparticles, when the amount of Ag seed added was (**a**) 0 µL, (**b**) 40 µL, (**c**) 80 µL, (**d**) 160 µL, and (**e**) 500 µL. (**f**) A statistical graph of the atomic ratio of Ag and Au in Ag@Au nanoparticles prepared with different amounts of silver seeds, according to the EDS results. (**g**) Schematic diagram of the growth process of Ag@Au nanoparticles. Scale bar: 100 nm.

Furthermore, we used the prepared Ag@Au nanoparticles as the substrate material for surface-enhanced Raman spectroscopy (SERS) for highly sensitive detection of the drug fentanyl. First, we explored the SERS enhancement effect of Ag@Au nanoparticles with different structures. Figure 4 shows the SERS spectra obtained with 10−<sup>4</sup> M *p*-aminothiophenol (4-ATP) as the test molecule and Ag@Au nanoparticles with different structures as the base material. We can see that the order of SERS enhancement effects, from high to low, is spherical Ag@Au nanoparticles, sea-urchin-shaped Ag@Au nanoparticles, and mesoporous gold nanoparticles. Therefore, in the follow-up test, we use spherical Ag@Au nanoparticles as the enhancement reagent. The Raman characteristic peaks of 4-ATP molecules are located at 1076 cm−<sup>1</sup> and 1585 cm−<sup>1</sup> [34], and we see obvious SERS peaks at 1140 cm−<sup>1</sup> , 1389 cm−<sup>1</sup> , and 1432 cm−<sup>1</sup> , which can be attributed to βCH + νCN, νNN + νCN, and νNN + βCH of *p*,*p*'-dimercaptoazobenzene (DMAB), respectively [4]. This indicates that the prepared nanomaterials have catalytic activity, so that 4-ATP is partially converted to DMAB by catalytic oxidation.

**Figure 4.** SERS spectra of 10−<sup>4</sup> M 4-ATP molecules with different substrate materials.

We used spherical Ag@Au nanoparticles to detect fentanyl. Figure 5a shows the Raman spectrum and SERS spectrum of the fentanyl standard substance. As shown in Figure 5a (black line), the Raman peaks of fentanyl solid powder are located at 621 cm−<sup>1</sup> , 746 cm−<sup>1</sup> , 831 cm−<sup>1</sup> , 1002 cm−<sup>1</sup> , 1030 cm−<sup>1</sup> , 1201 cm−<sup>1</sup> , 1447 cm−<sup>1</sup> , and 1600 cm−<sup>1</sup> , which is consistent with previous reports [35]. However, the peak at 621 cm−<sup>1</sup> in the SERS spectrum (red line) disappeared. This is because, for a group, the Raman peak of tensile vibration usually appears in a higher frequency range, and is less affected by the external environment. The Raman peak of deformation vibration is usually located in a lower frequency range, and is sensitive to environmental changes [36]. The peak at 621 cm−<sup>1</sup> is the bending vibration of C–C–C [35], which is easily affected by the SERS detection environment, and disappears. We assigned the SERS peak of fentanyl, as shown in Table A1. Furthermore we performed SERS tests on different concentrations of fentanyl, and the results are shown in Figure 5b. We can see that the lowest detection concentration is 10−<sup>7</sup> M.

**Figure 5.** (**a**) Normal and surface-enhanced Raman spectra for fentanyl (10−<sup>4</sup> M). (**b**) SERS spectra of fentanyl at different concentrations.
