Synthesis of Densely Immobilized Gold-Assembled Silica Nanostructures

In this study, dense gold-assembled SiO2 nanostructure (SiO2@Au) was successfully developed using the Au seed-mediated growth. First, SiO2 (150 nm) was prepared, modified by amino groups, and incubated by gold nanoparticles (ca. 3 nm Au metal nanoparticles (NPs)) to immobilize Au NPs to SiO2 surface. Then, Au NPs were grown on the prepared SiO2@Au seed by reducing chloroauric acid (HAuCl4) by ascorbic acid (AA) in the presence of polyvinylpyrrolidone (PVP). The presence of bigger (ca. 20 nm) Au NPs on the SiO2 surface was confirmed by transmittance electronic microscopy (TEM) images, color changes to dark blue, and UV-vis spectra broadening in the range of 450 to 750 nm. The SiO2@Au nanostructure showed several advantages compared to the hydrofluoric acid (HF)-treated SiO2@Au, such as easy separation, surface modification stability by 11-mercaptopundecanoic acid (R-COOH), 11-mercapto-1-undecanol (R-OH), and 1-undecanethiol (R-CH3), and a better peroxidase-like catalysis activity for 5,5′-Tetramethylbenzidine (TMB) and hydrogen peroxide (H2O2) reaction. The catalytic activity of SiO2@Au was two times better than that of HF-treated SiO2@Au. When SiO2@Au nanostructure was used as a surface enhanced Raman scattering (SERS) substrate, the signal of 4-aminophenol (4-ATP) on the surface of SiO2@Au was also stronger than that of HF-treated SiO2@Au. This study provides a potential method for nanoparticle preparation which can be replaced for Au NPs in further research and development.

Based on their dimensions, Au nanostructures are classified as one-dimensional (1D) nanostructures (nanorods, nanowires, nanotubes, nanobelts); two-dimensional (2D) nanostructures (nanoplates such as nanoparticles, stars, pentagons, squares/rectangles, dimpled nanoplates, hexagons, truncated triangles); and three-dimensional (3D) nanostructures (nanotadpoles, nanodumbbells, nanopods, nanostars, and nanodendrites) [23]. Even 2 of 12 though spherical or quasi-spherical Au NPs have received the most attention because of the ease of synthesis manipulation, anisotropically Au nanostructures exhibit unique physical and optical properties, such as low percolation threshold and surface plasmon resonance (SPR) [24]. In addition, the optical properties of anisotropic Au nanostructure such as the hollow nanoshell, and nanorods can be tunable with their shape in the visible region and in the near infrared (NIR) region. This property has given rise to the opportunity for using anisotropic Au nanostructures as composite therapeutic agents in clinical medical applications such as diagnostics and therapy [25]. However, the synthesis of higherdimensional Au nanostructures usually requires specific templates for guiding anisotropic growth [26]. Many templates have been utilized, including biomolecules (DNA), polymers, surfactants, inorganic nanowires, and lithographical patterns [27][28][29][30][31]. However, the synthesis of anisotropic Au nanostructures with precise control of morphology remains a great challenge.
Recently, our group reported Au-Ag alloys assembled SiO 2 (SiO 2 @Au-Ag) nanostructure as a strong and reliable surface enhanced Raman scattering (SERS) probe using SiO 2 as a template [32][33][34][35]. SiO 2 NPs are inert and their sizes are easily controllable [32]. Furthermore, the density of Au NPs, nanogaps, size and shape of nanostructure can be controlled by SiO 2 template, generating a homogenous SERS substrate [36]. The density of the Au NPs and the gaps between two Au NPs on the surface of SiO 2 can be tunable and provide a stronger localized surface plasmon resonance (LSPR) property. As a result, SiO 2 @Au-Ag can be developed as high-strength and reliable SERS substrates for the enzyme-linked immunosorbent assay (ELISA) [37,38], which is an internal SERS [34,35], pesticide detection standard [39], and provides detection of toxic chemical compound in pharmaceutics [40]. However, the cellular toxicity and easy oxidation of intrinsic Ag of our nanostructure limits their application in vivo [41,42]. Even though some research groups have investigated the attachment of Au NP on the surface of SiO 2 , the particle density on the SiO 2 surface was nearly low or non-uniform [43][44][45][46][47][48]. Therefore, we used the combination of SiO 2 core template and seed-growth methods in this study to develop a new Au-based SiO 2 nanostructure with dense Au density on the surface of SiO 2 . This study provides a potential method for preparation of nanoparticles including noble metal that can be replaced for spherical Au NPs, which supports further research and development.

Results and Discussion
To prepare SiO 2 nanostructure (SiO 2 @Au), silica NPs (~150 nm) were first functionalized by 3-aminopropyltriethoxysilane (APTS) to prepare aminated silica NPs, as shown in Figure 1a. Simultaneously, colloidal Au NPs (3 nm) were synthesized by tetrakis(hydroxymethyl)phosphonium chloride (THPC) and incubated with the aminated silica NPs to prepare Au NPs seed embedded with SiO 2 (SiO 2 @Au seed NPs), according to the method reported by Pham et al. [32][33][34][35]39]. Subsequently, the Au NPs on the surface of SiO 2 @Au seed were grown by reducing a gold precursor (HAuCl 4 ) in the presence of ascorbic acid (AA) and polyvinylpyrrolidone (PVP) as a stabilizer and structure-directing agent under mild reducing conditions [32]. The gold ions reduced by AA were selectively grown onto SiO 2 @Au seed to increase the size of Au on the surface of SiO 2 NPs to generate SiO 2 @Au.

Synthesis of SiO2@Au Nanostructure
We investigated the characteristics of SiO2@Au synthesized by reduction of HAuCl4 and AA in the presence of PVP. Figure 1b shows the optical images of nanostructures in our research. SiO2 NPs with ca. 150 nm in diameter are shown in a transparent solution in Figure 1bi. When Au NPs were coated and grown on the surface of aminated SiO2, the color of the solution changed to light brown, as shown in Figure 1bii. Then, 2, 5, and 10 mg of SiO2@NH2 were incubated in 10 mL Au NPs suspension overnight ( Figure S1a). The surface of SiO2 NPs was decorated with many small Au NPs to generate the SiO2@Au seed ( Figure 1bii and Figure S1a). The Au NPs density on the SiO2 surface became inversed with the amount of SiO2 used. Then, 1 mg of SiO2@Au seed was used for a growth of Au NPs on the SiO2 in Figure S1b-d. The sizes of Au NPs seed on the SiO2@Au surface increased with the Au 3+ concentration in the range of 100 to 300 µM at all 2, 5, and 10 mg SiO2@NH2 ( Figure S1b-d). The ultraviolet-visible (UV-Vis) spectra of obtained SiO2@Au nanostructures synthesized at different concentrations of Au 3+ were investigated in Figure S1e. The optical property of SiO2@Au was slightly tunable by adjusting the Au 3+ concentration in the range of 100 to 300 µM. At 100 µM Au 3+ , the maximum absorbance peaks of obtained SiO2@Au synthesized by 2, 5, and 10 mg SiO2@NH2 were observed at 523, 522, and 521 nm, respectively. Whereas these peaks were red-shifted to 547, 543, and 541 nm at 200 µM Au 3+ and 571, 561, and 572 nm at 300 µM Au 3+ , respectively. These results were consistent with the Mie's theory which states that an increase of particle size leads the electric surface charge density and shifts the plasmon absorption band to longer wavelengths [23]. However, it is difficult to obtain the different desired SiO2@Au

Synthesis of SiO 2 @Au Nanostructure
We investigated the characteristics of SiO 2 @Au synthesized by reduction of HAuCl 4 and AA in the presence of PVP. Figure 1b shows the optical images of nanostructures in our research. SiO 2 NPs with ca. 150 nm in diameter are shown in a transparent solution in Figure 1b(i). When Au NPs were coated and grown on the surface of aminated SiO 2 , the color of the solution changed to light brown, as shown in Figure 1b(ii). Then, 2, 5, and 10 mg of SiO 2 @NH 2 were incubated in 10 mL Au NPs suspension overnight ( Figure S1a). The surface of SiO 2 NPs was decorated with many small Au NPs to generate the SiO 2 @Au seed (Figure 1b(ii) and Figure S1a). The Au NPs density on the SiO 2 surface became inversed with the amount of SiO 2 used. Then, 1 mg of SiO 2 @Au seed was used for a growth of Au NPs on the SiO 2 in Figure S1b-d. The sizes of Au NPs seed on the SiO 2 @Au surface increased with the Au 3+ concentration in the range of 100 to 300 µM at all 2, 5, and 10 mg SiO 2 @NH 2 ( Figure S1b-d). The ultraviolet-visible (UV-Vis) spectra of obtained SiO 2 @Au nanostructures synthesized at different concentrations of Au 3+ were investigated in Figure  S1e. The optical property of SiO 2 @Au was slightly tunable by adjusting the Au 3+ concentration in the range of 100 to 300 µM. At 100 µM Au 3+ , the maximum absorbance peaks of obtained SiO 2 @Au synthesized by 2, 5, and 10 mg SiO 2 @NH 2 were observed at 523, 522, and 521 nm, respectively. Whereas these peaks were red-shifted to 547, 543, and 541 nm at 200 µM Au 3+ and 571, 561, and 572 nm at 300 µM Au 3+ , respectively. These results were consistent with the Mie's theory which states that an increase of particle size leads the electric surface charge density and shifts the plasmon absorption band to longer wavelengths [23]. However, it is difficult to obtain the different desired SiO 2 @Au nanostructures as expected when using 1 mg SiO 2 @Au seed. The final size of Au NPs is dependent on the number of seeds and total concentration of Au 3+ in the growth solution [49]. In our study, the Au 3+ concentration in the growth solution was fixed at 100, 200, and 300 µM. Therefore, the final size of Au NPs on the surface of SiO 2 depended on the number of SiO 2 @Au seeds (or SiO 2 @Au seed amount). Indeed, 1 mg of SiO 2 @Au seed was used for Au NPs growth process in our study, which resulted in Au NPs on the SiO 2 surface not being able to grow larger as seen in the TEM images and UV-Vis spectra ( Figure S1). To obtain a monodisperse SiO 2 @Au nanostructure, the SiO 2 @Au seed was therefore decreased to 0.2 mg for the growth of Au NPs on the SiO 2 NPs in the next study. When 0.2 mg SiO 2 @Au seed was used as seed, the color of the obtained SiO 2 @Au suspension obviously changed from light brown to dark blue when the Au 3+ concentration increased to 150 µM, as shown in Figure 1b(iii). According to the literature, Au NPs exhibit color and LSPR bands in the visible light that are dependent on size and shape [49][50][51][52]. Any changes in the absorbance or wavelength of the LSPR band provide a difference in size, shape, and aggregation state. This result indicated that the growth of Au NPs on the SiO 2 surface was well performed and the absorbance, transmission, and reflection of light passed through the SiO 2 @Au suspension were also different at various synthesis conditions of SiO 2 @Au nanostructures. The UV-Vis spectra of the SiO 2 @Au nanostructures synthesized at 150 µM Au 3+ were investigated in Figure 1c to show additional characteristics of SiO 2 @Au nanostructures. The suspension of SiO 2 and SiO 2 @Au seed did not show any absorbance in the range of 300-1000 nm because of its small Au NPs on the SiO 2 surface [37,53]. Whereas the suspension of SiO 2 @Au synthesized at 150 µM Au 3+ showed a broadband in the range of 450 to 750 nm with the maximum peak at 615 nm. Furthermore, the absorbance intensity of SiO 2 @Au nanostructure solution was sharply increased. Consistent with the UV-Vis spectra, the transmittance electronic microscopy (TEM) images of SiO 2 @Au nanostructures also showed that the size of Au NPs on the surface of SiO 2 @Au increased dramatically comparing to the SiO 2 @Au seed at 150 µM Au 3+ (Figure 1d), which led to the sharp broadening of LSPR bands of UV-Vis spectrum. While the average size of SiO 2 @Au seed was 200 ± 9 nm (n = 170, Figure S2a), the average size of SiO 2 @Au was 211 ± 7 nm (n = 172, Figure S2b). Consequently, the red-shifted and broadened absorbance properties of SiO 2 @Au nanostructures indicated the growth of Au NPs to bigger size on the SiO 2 surface [23,49]. Therefore, the SiO 2 @Au nanostructure synthesized at 150 µM Au 3+ was used for further study.

Characteristics of SiO 2 @Au Nanostructure
As shown in the previous study, the presence of SiO 2 template in SiO 2 -based nanostructure facilitates the control of density, size, shape, and nanogap of metal NPs on the SiO 2 surface [36]. It is believed that SiO 2 template also facilitates the separation of SiO 2based nanostructures in reaction compared to those without SiO 2 template. In our study, SiO 2 @Au nanostructure was first obtained from the growth reaction at 150 µM Au 3+ , then was incubated in 24% hydrofluoric acid (HF) to etch the SiO 2 core template as seen in Figure 2a. Figure 2b shows an obvious color change from blue to pink when the SiO 2 template was etched by HF. The TEM images of SiO 2 @Au nanostructure with HF treatment were collected. Figure 1d(iii) shows the morphology of SiO 2 @Au without HF treatment with the average size of 211 ± 7 nm ( Figure S2b). Many Au NPs presented on the surface of SiO 2 template. However, the nanostructure with HF treatment showed many small Au NPs with different shape (Figure 2c(ii)). The average size of Au NPs was 7.7 ± 1.5 nm (n = 172, Figure S2c). This meant that SiO 2 core was completely etched and disappeared in SiO 2 @Au nanostructure and remained as small Au NPs. The optical properties of SiO 2 @Au with and without HF treatment (Au) were significantly different, as shown in Figure 2d. Both the maximum peak position and absorbance intensity of SiO 2 @Au with HF treatment (Au) decreased dramatically. The maximum peak position of SiO 2 @Au at 615 nm was blue-shifted to 546 nm when SiO 2 core was etched. The difference in the LSPR wavelength and absorbance intensity of Au (SiO 2 @Au with HF treatment) was due to the difference in aggregation state of nanostructure that was caused by the disappearance of SiO 2 core template [49]. To confirm the aggregation state of SiO 2 @Au with and without SiO 2 core, the nanostructures were redispersed in 1 M NaCl. The maximum LSPR peak of SiO 2 @Au in NaCl showed a 10 nm red-shift compared to it in distilled water. Whereas SiO 2 @Au treated with HF was 22 nm red-shifted in 1 M NaCl ( Figure S3). This indicates that SiO 2 @Au treated with HF was more easily aggregated than SiO 2 @Au without HF treatment. treatment) was due to the difference in aggregation state of nanostructure that was caused by the disappearance of SiO2 core template [49]. To confirm the aggregation state of SiO2@Au with and without SiO2 core, the nanostructures were redispersed in 1 M NaCl. The maximum LSPR peak of SiO2@Au in NaCl showed a 10 nm red-shift compared to it in distilled water. Whereas SiO2@Au treated with HF was 22 nm red-shifted in 1 M NaCl ( Figure S3). This indicates that SiO2@Au treated with HF was more easily aggregated than SiO2@Au without HF treatment. The separation and redispersion of SiO2@Au nanostructures was also observed in Figure 2f,g and Figure S3b-d. The SiO2@Au with and without HF treatment were centrifuged at various centrifugation speeds in the range of 1000 to 18,000× g. The SiO2@Au was almost settled down to a bottle of microtube at a centrifugal speed of 3000 g and left a supernatant which showed a low absorbance. Meanwhile, the absorbance intensity of the supernatant of SiO2@Au treated with HF decreased gradually to 50% at 1000× g, 25% at 3000 g and 6.3% at 10,000 g. From these results, we concluded that SiO2@Au was easily centrifuged and separated from the reaction solution. In the next experiment, we carried out centrifuge and successively redispersed the SiO2@Au at the centrifugation speed of 3000 g. As seen in Figure 2g and Figure S3c, both SiO2@Au with and without HF treatment was easily redispersed in phosphate buffer saline containing 0.1% Tween 20 (PBST). Therefore, we concluded that the SiO2@Au nanostructures were easily separated and redispersed in PBST at mild conditions. The use of SiO2 as the core template opens up a new opportunity for easy preparation and separation of the nanostructure.

Surface Modification of SiO2@Au Nanostructure
According to the literature, the electric field of the incident light induces polarization and excites the free conduction electron on the surface of the nanostructure to gen- The separation and redispersion of SiO 2 @Au nanostructures was also observed in Figure 2f,g and Figure S3b-d. The SiO 2 @Au with and without HF treatment were centrifuged at various centrifugation speeds in the range of 1000 to 18,000× g. The SiO 2 @Au was almost settled down to a bottle of microtube at a centrifugal speed of 3000× g and left a supernatant which showed a low absorbance. Meanwhile, the absorbance intensity of the supernatant of SiO 2 @Au treated with HF decreased gradually to 50% at 1000× g, 25% at 3000× g and 6.3% at 10,000× g. From these results, we concluded that SiO 2 @Au was easily centrifuged and separated from the reaction solution. In the next experiment, we carried out centrifuge and successively redispersed the SiO 2 @Au at the centrifugation speed of 3000× g. As seen in Figure 2g and Figure S3c, both SiO 2 @Au with and without HF treatment was easily redispersed in phosphate buffer saline containing 0.1% Tween 20 (PBST). Therefore, we concluded that the SiO 2 @Au nanostructures were easily separated and redispersed in PBST at mild conditions. The use of SiO 2 as the core template opens up a new opportunity for easy preparation and separation of the nanostructure.

Surface Modification of SiO 2 @Au Nanostructure
According to the literature, the electric field of the incident light induces polarization and excites the free conduction electron on the surface of the nanostructure to generate the LSPR spectrum. Therefore, any variation in the LSPR absorbance intensity or wavelength represents the difference in particle size, shape, aggregation state, as well as free electron cloud on the surface of the nanostructure [49,52]. We used the UV-Vis spectrum to observe the optical properties of the SiO 2 @Au nanostructures modified by three kinds of ligand (11-mercaptopundecanoic acid (R-COOH), 11-mercapto-1-undecanol (R-OH), and 1-undecanethiol (R-CH 3 )). Similarly, SiO 2 @Au which removed the SiO 2 core by HF treatment was also used as a control sample to compare its optical properties to SiO 2 @Au nanostructures. The results are shown in Figure S4 and Figure 3. When R-COOH, R-CH 3 and R-OH ligands were modified on the SiO 2 @Au surface, the absorbance intensities of all prepared SiO 2 @Au nanostructures obviously decreased ( Figure S4). In addition, the extinction peak positions of SiO 2 @Au nanostructures modified by ligands were all blueshifts (Figure 3a(i)). Maximum peak of SiO 2 @Au nanostructures modified by R-COOH ligand dramatically decreased 50 nm from 615 nm to 557 nm. Similarly, the SiO 2 @Au nanostructures modified by R-OH ligand also showed a 75 nm blue-shift to 540 nm and those modified by R-CH 3 showed a 42 nm blue-shift to 573 nm, respectively (Figure 3b(i)). Meanwhile, the HF-treated SiO 2 @Au modified by R-COOH, R-OH, and R-CH 3 ligands exhibited different behaviors (Figure 3a(ii)). Even though the extinction peak positions of the HF-treated SiO 2 @Au modified by R-COOH and R-OH also showed decreases in LSPR wavelength like those of SiO 2 @Au, the blue-shift differences in wavelength were small, just 15 and 18 nm for R-COOH and R-OH ligands, respectively (Figure 3b(ii)). Blue-shift decreases in wavelength of SiO 2 @Au nanostructures modified by R-COOH and R-OH are due to hydrophilic properties of OH and COOH groups of the modified nanostructure regardless of HF treatment. Interestingly, the HF-treated SiO 2 @Au modified by R-CH 3 showed an opposite behavior compared to that of SiO 2 @Au nanostructure. While the SiO 2 @Au nanostructure modified by R-CH 3 showed a blue-shift from 615 nm to 573 nm, the HF-treated SiO 2 @Au nanostructure modified by R-CH 3 showed a red-shift from 546 to 561 nm. This indicates the presence of aggregation of the HF-treated SiO 2 @Au nanostructure modified by R-CH 3 . In contrast, presence of R-CH 3 groups on the surface of SiO 2 @Au nanostructures showed little effect on the optical property of SiO 2 @Au nanostructure because of the big size of SiO 2 @Au (~211 nm) compared to HF-treated SiO 2 @Au (~8 nm). This is because of the intrinsic hydrophobic property of CH 3 group strongly present on the surface of small Au, as shown in Figure 2c (~8 nm). The result was confirmed by replacing R-CH 3 group by 4-aminophenol (4-ATP) with a benzene ring. Figure S5a,b show that the extinction bands of both SiO 2 @Au with and without HF treatment were red-shifted because of the intrinsic hydrophobic aromatic ring of 4-ATP. From these results, we once again concluded that the SiO 2 @Au nanostructure possessed a stable surface for ligand modification compared to that of Au NPs without the SiO 2 core.

SERS and Peroxidase-Like Activity of SiO 2 @Au Nanostructure
The application of the SiO 2 @Au nanostructure was briefly performed in our study. In SERS measurement, we used 4-ATP as a Raman reporter to observe the SERS properties of SiO 2 @Au with and without HF treatment using a 780 nm diode pump solid-state laser. Figure 4a shows the SERS spectra of SiO 2 @Au and HF-treated SiO 2 @Au before and after incubating in 1 mM 4-ATP solution. Although several Raman signals were shown in 4-ATP untreated condition, after incubating in 1 mM 4-ATP solution, typical bands of 4-ATP were clearly obtained for both SiO 2 @Au and HF-treated SiO 2 @Au due to the electromagnetic enhancement of the decorated Au NPs. Dominant and distinct bands were seen at 1081 cm −1 which were assigned to C-H in-plane bending vibration. The band at 1592 cm −1 and 391 cm −1 were attributed to C-C ring and C-S stretching, respectively [32]. Interestingly, the SERS bands at 391, 1081, and 1592 cm −1 of SiO 2 @Au were stronger than those of HF-treated SiO 2 @Au due to the hot spot generated by Au NPs on the surface of SiO 2 @Au. These results demonstrate the importance of pre-seeding Au NPs to ensure the growth of Au NPs onto the SiO 2 surface to generate various hot spots, enhance the electromagnetic field on or near the surface of SiO 2 @Au, and amplify the SERS signal of SiO 2 @Au compared to HF-treated SiO 2 @Au without the SiO 2 core template.

SERS and Peroxidase-like Activity of SiO2@Au Nanostructure
The application of the SiO2@Au nanostructure was briefly performed in our st In SERS measurement, we used 4-ATP as a Raman reporter to observe the SERS pro ties of SiO2@Au with and without HF treatment using a 780 nm diode pump solidlaser. Figure 4a shows the SERS spectra of SiO2@Au and HF-treated SiO2@Au before after incubating in 1 mM 4-ATP solution. Although several Raman signals were show 4-ATP untreated condition, after incubating in 1 mM 4-ATP solution, typical ban 4-ATP were clearly obtained for both SiO2@Au and HF-treated SiO2@Au due to the tromagnetic enhancement of the decorated Au NPs. Dominant and distinct bands seen at 1081 cm -1 which were assigned to C-H in-plane bending vibration. The ban 1592 cm -1 and 391 cm -1 were attributed to C-C ring and C-S stretching, respectively Interestingly, the SERS bands at 391, 1081, and 1592 cm −1 of SiO2@Au were stronger those of HF-treated SiO2@Au due to the hot spot generated by Au NPs on the surfa SiO2@Au. These results demonstrate the importance of pre-seeding Au NPs to ensur growth of Au NPs onto the SiO2 surface to generate various hot spots, enhance the tromagnetic field on or near the surface of SiO2@Au, and amplify the SERS sign SiO2@Au compared to HF-treated SiO2@Au without the SiO2 core template.  In addition, Au NPs-based nanostructures showed peroxidase-like catalytic activities in previous reports [54][55][56]. In this study, we used the SiO2@Au nanostructure to catalyze for 5,5'-Tetramethylbenzidine (TMB) and hydrogen peroxide (H2O2) reaction. Figure 4b shows the UV-Vis spectra of the SiO2@Au with and without HF treatment. In the presence of H2O2 and under the catalysis of nanostructures, TMB was oxidized to TMB + , followed by conversion to TMB 2+ in sulfuric acid (H2SO4) condition as mentioned In addition, Au NPs-based nanostructures showed peroxidase-like catalytic activities in previous reports [54][55][56]. In this study, we used the SiO 2 @Au nanostructure to catalyze for 5,5 -Tetramethylbenzidine (TMB) and hydrogen peroxide (H 2 O 2 ) reaction. Figure 4b shows the UV-Vis spectra of the SiO 2 @Au with and without HF treatment. In the presence of H 2 O 2 and under the catalysis of nanostructures, TMB was oxidized to TMB + , followed by conversion to TMB 2+ in sulfuric acid (H 2 SO4) condition as mentioned in Figure S4c. The presence of TMB 2+ in reaction will show a clear absorbance peak at~450 nm. Indeed, TMB was successfully converted to TMB 2+ under the catalysis of the SiO 2 @Au by the presence of a clear and strong absorbance band at 453 nm (Figure 4b(i)). Similarly, the HF-treated SiO 2 @Au nanostructures could also convert TMB to TMB 2+ , as shown in in Figure 4b(ii). However, the absorbance intensity at 453 nm of the SiO 2 @Au was two times higher than that of the SiO 2 @Au with HF treatment. This is perhaps because of the easy aggregation of the HF-treated SiO 2 @Au nanostructure or because the surface of Au NPs was varied in the HF treatment as mentioned in a previous report [54].

Preparation of SiO 2 @Au NPs
The SiO 2 @Au seed was prepared as in the previous report [32,33]. The SiO 2 @Au NPs were prepared by incubating Au NPs suspension (10 mL) with aminated silica NPs (2 mg) overnight. The colloids were carefully centrifuged and washed thoroughly using EtOH. The NPs were then redispersed in 2.0 mL of 1 mg/mL PVP of obtained 1 mg/mL SiO 2 @Au seed.
The SiO 2 @Au NPs were carefully prepared via the reduction and deposition of Au using AA onto SiO 2 @Au seed in PVP. Moreover, 200 µL of SiO 2 @Au (1.0 mg/mL) was briefly dispersed in 9.8 mL of water that contained 10 mg of PVP under stirring. Thereafter, 20 µL of HAuCl 4 (10 mM) was added to the suspension, followed by the addition of 40 µL of AA (10 mM). The suspension was incubated for 15 min to completely reduce the Au 3+ ions to Au 0 . By repeating the reduction steps, the final Au 3+ concentration was controlled to be 150 µM. The SiO 2 @Au NPs were obtained by the centrifugation of the suspension at 8500 rpm for 15 min, then the NPs were washed thoroughly using EtOH to remove any excess reagent. The SiO 2 @Au NPs were then redispersed in 1 mL of absolute EtOH to obtain a 200 µg/mL SiO 2 @Au NP suspension.

Etching of Silica Core of SiO 2 @Au NPs
In order to etch the silica core of SiO 2 @Au, 500 µL of SiO 2 @Au (100 µg/mL) in PBS solution which contained 0.1% Tween 20 (PBST), was added in 500 µL of 48% HF, followed by incubation for 24 h and centrifugation for 15 min at 17,000 rpm to obtain the Au NPs suspension. The prepared Au NPs were washed thoroughly using PBST to remove any excess reagent. The Au NPs were then redispersed in 1 mL of PBST, to obtain a 50 µg/mL Au NPs suspension.

Separation and Dispersion of SiO 2 @Au
First, 50 µg/mL SiO 2 @Au suspension was prepared in PBST and centrifuged for 10 min at various centrifugation speeds in the range of 1000 to 18,000× g. The supernatant was collected and the UV-Vis spectroscopy measured in the range of 300-800 nm. The pellet was redispersed by sonication. The centrifugation and redispersion were repeated until finishing. Similarly, the separation and redispersion of 50 µg/mL Au suspension was carried out as SiO 2 @Au suspension.

Surface Modification of SiO 2 @Au
SiO 2 @Au was incubated with 1 mM ligands, such as 1-undecanethiol (R-CH 3 ), 11mercaptopundecanoic acid (R-COOH), and 11-mercapto-1-undecanol (R-OH) for 6 h at room temperature to modify the surface of Au NPs by CH 3 , COOH, and OH groups, respectively. Briefly, 50 µg SiO 2 @Au suspension was prepared in 500 µL EtOH. Then, 2 mM ligand solutions (500 µL) were added into SiO 2 @Au suspension and incubated for 6 h under stirring. The nanostructure was collected at 17,000 rpm for 15 min and the obtained pellet was redispersed in 1000 µL PBST to obtain an SiO 2 @Au-ligand suspension. Similarly, the surface modification of Au NPs was carried out as SiO 2 @Au NPs.

Peroxidase-like Catalytic Activity of SiO 2 @Au
In order to obtain the peroxidase-like catalysis of nanostructures, TMB was dissolved in EtOH to obtain a stock solution of 10 mM TMB. Similarly, a stock solution of 2 M H 2 O 2 was freshly prepared. Thereafter, 100 µL of 10 mM TMB, 700 µL of buffer pH 4.0, 100 µL of 2 M H 2 O 2 , and 100 µL of nanostructure (200 µg/mL) were incubated for 30 min at 25 • C in vortex mixer. The mixture was added to 500 µL of 1 M H 2 SO4 to stop the reaction and incubated for 10 min for color development. The suspension was measured by UV-Vis spectroscopy at 453 nm.

Instrument
Tranmission electron microscope images of the sample were captured by using Libra 120 field-emission transmission electron microscope (Carl Zeiss, Oberkochen, Baden-Württemberg, Germany) with a maximum accelerated voltage of 120 kV and JEM-F200 multi-purpose electron microscope (JEOL, Akishima, Tokyo, Japan) with a maximum accelerated voltage of 200 kV. Optical properties of the sample were observed by an Optizen POP UV/Vis spectrometer (Mecasys, Seoul, Korea). Centrifugation of the sample was performed by using a Microcentrifuge 1730R (LaboGene, Lyngen, Denmark). The Raman signal of nanostructure suspension in the capillary tube was recorded by a micro-Raman system (LabRam 300, JY-Horiba, Tokyo, Japan) equipped with an optical microscope (BX41, Olympus, Tokyo, Japan). The SERS signals were collected with a back-scattering geometry using a ×10 objective lens (0.90 NA, Olympus) and with a spectrometer using a thermoelectrically cooled CCD detector. A 780 nm diode-pumped solid-state laser (CL780-100-S, CrystaLaser, Reno, NV, USA) was used as a photoexcitation source with 50 mW of laser power shot at the sample.

Conclusions
Dense Au NPs on the surface of SiO 2 @Au was successfully developed using the combination of Au seed-mediated growth in this study. The growth of Au NPs on the surface of SiO 2 was confirmed by the color changes from light brown to dark blue, TEM images, and broad bands in the UV-vis spectra in the range of 450 nm to 750 nm with the maximum peak at 615 nm, which indicated bigger size of Au NPs on SiO 2 surface when the Au 3+ concentration increased to 150 µM. In addition, the prepared SiO 2 @Au nanostructure showed several advantages compared to the SiO 2 @Au treated by HF solution, such as easy separation from the solution at 3000× g for 10 min and stability for surface modification by R-COOH, R-OH, and R-CH 3 . Furthermore, the SiO 2 @Au nanostructure was used as a SERS substrate for 4-ATP detection and a peroxidase-like catalyst material for TMB and H 2 O 2 reactions. As a result, the SiO 2 @Au showed a stronger SERS signal of SiO 2 @Au compared to HF-treated SiO 2 @Au. Similarly, the catalytic activity of SiO 2 @Au was two times better than that of the SiO 2 @Au with HF treatment. This study provides a potential method for nanoparticle preparation that can be replaced for Au NPs, which supports further research and development.