Synthesis of Finely Controllable Sizes of Au Nanoparticles on a Silica Template and Their Nanozyme Properties

The precise synthesis of fine-sized nanoparticles is critical for realizing the advantages of nanoparticles for various applications. We developed a technique for preparing finely controllable sizes of gold nanoparticles (Au NPs) on a silica template, using the seed-mediated growth and interval dropping methods. These Au NPs, embedded on silica nanospheres (SiO2@Au NPs), possess peroxidase-like activity as nanozymes and have several advantages over other nanoparticle-based nanozymes. We confirmed their peroxidase activity; in addition, factors affecting the activity were investigated by varying the reaction conditions, such as concentrations of tetramethyl benzidine and H2O2, pH, particle amount, reaction time, and termination time. We found that SiO2@Au NPs are highly stable under long-term storage and reusable for five cycles. Our study, therefore, provides a novel method for controlling the properties of nanoparticles and for developing nanoparticle-based nanozymes.


Introduction
Enzymes are biocatalysts that play an important role in living systems. However, they are expensive, difficult to store, laborious to produce, and are easily denatured in external environments from varying temperature, pH, and chemical stressors [1,2]. These drawbacks critically limit their practical uses [3].
Among the various metal nanomaterials, Au NPs have attracted the most attention because of their outstanding catalytic properties and advantages [23][24][25][26][27][28][29][30][31][32][33][34][35]. Au NPs can be synthesized easily and are stable [36,37]. In addition, the physical/chemical properties of Au NPs can be controlled by controlling their size and shape [24,26,33,35]. These NPs are also highly biocompatible and are easy to functionalize [24,30,35,[38][39][40][41][42]. However, gold is 2 of 13 not cheap, and the catalytic reactions depend on the surface area of the NPs. Fine-sized NPs can be cost-effective, but they are difficult to separate for reuse [1,3,[8][9][10]17,43,44]. The development of a structure that uses small amounts of gold to acquire a large surface area, while still being easily separated, might prove to be a highly useful material in the field of nanozymes. A nanostructure where Au NPs are assembled onto a silica (SiO 2 ) sphere core was developed by the Halas group in 1998 [45]. The chemical and optical properties of the Au NP-embedded SiO 2 structure can be changed by simply controlling the diameter of the core and layered nanoparticles [46][47][48][49][50][51]. The NP-embedded SiO 2 nanostructures are also cost-effective since only small amounts of expensive Au NPs are embedded onto the silica core, and they are easily separated from the reaction solution with the SiO 2 core.
Au NP-assembled SiO 2 nanostructures have been investigated in various fields [50,[52][53][54][55][56] and have been found to have many merits due to the combined properties of Au NPs and of the silica core, simultaneously utilizing the outstanding and unique features of Au NPs and the inert and versatile feature of SiO 2 [52,54,55,57]. In addition, the absorbance spectra of the nanostructures can be tuned across the visible and infrared regions by controlling the size of the Au NPs [38,39,58,59]. Due to these properties, the Au NP-assembled SiO 2 nanostructures have broad applications. While there are many possible approaches for the control of the size and density of Au NPs on a template surface, a method of synthesis for those nanostructures has not yet been established [46,[48][49][50]58,59]. For this reason, the low density and non-uniform morphology of Au NPs on nanostructures remain a considerable challenge [46,[48][49][50]. There is, therefore, a need for the development of an improved method for preparing Au-NP-assembled silica nanostructures.
Our group recently developed SiO 2 @Au nanostructures in which Au NPs were densely immobilized on the surface of a SiO 2 nanosphere [60]. For this nanostructure, the SiO 2 nanosphere was used as a template, and the Au NPs were uniformly and densely introduced on it, using the seed-mediated growth method. SiO 2 @Au nanostructures have enhanced separation and re-dispersion properties and are more stable during surface modification than Au NPs. Moreover, they have shown potential as effective nanozymes. In this study, besides introducing dense and uniform Au NPs onto the SiO 2 nanospheres, we also developed a facile method to very precisely control the size of Au NPs on the SiO 2 surface, and we investigated their optical and catalytic characteristics by controlling the size of the Au NPs. Furthermore, various factors affecting the peroxidase-like activity of SiO 2 @Au NPs were also studied.

Preparation of Size-Controlled Au NPs-Assembled Silica Nanostructures (SiO 2 @Au NPs)
The SiO 2 @Au NPs were prepared by using seed-mediated growth synthesis, consisting of two steps: embedding Au seeds on the SiO 2 surface, and growth of the Au NPs via the addition of an Au 3+ precursor and reductant in intervals [45,52]. First, the Au seeds (~2.5 nm) were prepared by using an Au 3+ precursor (HAuCl 4 ) and tetrakis(hydroxymethyl)phosphonium chloride (THPC). Then, the Au seeds were incubated with aminated SiO 2 nanospheres (~160 nm) to obtain Au-seeded SiO 2 nanospheres, as previously reported [56,57,[60][61][62]. On the Au-seeded SiO 2 nanospheres, the reduction of the Au 3+ precursor was directly conducted by using ascorbic acid (AA), which is a mild reducing agent, in the presence of polyvinylpyrrolidone (PVP) as a stabilizer. In these mildly reducing conditions, the progress of the growth stage is much slower than in strongly reducing conditions, making it easier to control the growth procedure [36]. A low concentration of the Au 3+ precursor and AA were added onto the Au-seeded SiO 2 in 5 min intervals until the desired concentrations for fine control over the size of the Au NPs are attained. The amounts are indicated in Table 1.   Figure 1.  First, numerous small Au NPs (~2.5 nm) were attached throughout the SiO 2 surface as seeds for the further growth of Au NPs. It is called SiO 2 @Au NP without the Au (III) precursor (0 µM), as shown in Figure 1b(i).
Subsequently, various concentrations of the Au 3+ precursor were added to the Auseeded SiO 2 nanospheres at intervals; the size of the Au NPs grew larger as the Au 3+ concentration was increased in Figure 1b(ii-vi). Moreover, the size of the Au NPs on the SiO 2 nanospheres was precisely controlled with high levels of density and uniformity, which were confirmed clearly, as shown in Figure 1. At a high concentration of Au 3+ (>200 µM), Au NPs on the SiO 2 surface merged with each other and became one larger particle. When the size of the Au NPs increased, the color of the solution changed in the order of pale brown, then pink, purple, dark blue, and finally black (Figure 2a). These changes occurred depending on the size and shape of the NPs, due to their localized surface plasmon resonance (LSPR) [37,46,48]. When nanoparticles are close to one another, the absorption spectra of proximally located nanoparticles red-shift considerably from that of solitary particles [58,59]. Increasing the space between the particles reduces the shift [59]. Mie's theory accounts for how increasing nanoparticle diameters induces the absorption spectra to red-shift by changing the electric surface charge density of the NPs [58]. The results of our study showed that the growth of Au NPs on SiO 2 @Au was controlled well by the color change of the particle suspension, transmission electron microscopy (TEM) images, and absorbance according to the abovementioned theories. Subsequently, various concentrations of the Au 3+ precursor were added to the Auseeded SiO2 nanospheres at intervals; the size of the Au NPs grew larger as the Au 3+ concentration was increased in Figure 1b(ii-vi). Moreover, the size of the Au NPs on the SiO2 nanospheres was precisely controlled with high levels of density and uniformity, which were confirmed clearly, as shown in Figure 1. At a high concentration of Au 3+ (>200 µM), Au NPs on the SiO2 surface merged with each other and became one larger particle. When the size of the Au NPs increased, the color of the solution changed in the order of pale brown, then pink, purple, dark blue, and finally black (Figure 2a). These changes occurred depending on the size and shape of the NPs, due to their localized surface plasmon resonance (LSPR) [37,46,48]. When nanoparticles are close to one another, the absorption spectra of proximally located nanoparticles red-shift considerably from that of solitary particles [58,59]. Increasing the space between the particles reduces the shift [59]. Mie's theory accounts for how increasing nanoparticle diameters induces the absorption spectra to red-shift by changing the electric surface charge density of the NPs [58]. The results of our study showed that the growth of Au NPs on SiO2@Au was controlled well by the color change of the particle suspension, transmission electron microscopy (TEM) images, and absorbance according to the abovementioned theories. The absorption spectra of SiO2 showed that the absorbance band was red-shifted and broadened upon an increase in the concentration of the Au 3+ precursor, indicating the formation of larger Au NPs, which change the proximate interparticle distance (Figure 2b,c). In the absorbance band of SiO2@Au-NPs-treated 0 µM Au 3+ precursor, no peak was observed due to the exceedingly tiny size of the Au NPs. On the other hand, 50, 100, 150, 200, and 300 Au 3+ -treated SiO2@Au showed peaks at 543, 571, 593, 619, and 632 nm respectively ( Figure 2c). Moreover, the absorbance bandwidth broadened as the size of the Au NPs was increased.

Verification of the Peroxidase-like Activity of SiO2@Au NPs
The peroxidase-like activity of SiO2@Au NPs was evaluated through oxidation of a 3,3′,5,5′-tetramethylbenzidine (TMB) substrate. The TMB oxidation reaction involves the transfer of two electrons that each produce a clear color change. When the first electron is transferred to form TMB + via oxidization of TMB, the TMB solution changes from colorless to blue. Since TMB + is quite unstable in an acidic environment, it further oxidizes to TMB 2+ when the second electron is transferred; TMB 2+ is stable in acidic conditions, exhibiting a yellow color and a maximum absorption peak at 453 nm ( Figure 3a) [63].
To confirm the peroxidase-like activity of the SiO2@Au NPs, TMB + H2O2, TMB + SiO2@Au NPs, and TMB + H2O2 + SiO2@Au NPs were prepared in a pH 4 buffer for the peroxidase assay. The TMB + H2O2 solution was colorless, and an absorbance peak at 453 nm did not appear, as shown in Figure 3b,c. This result indicated that peroxidase-like activity did not occur in the absence of SiO2@Au. Next, the color of the TMB + SiO2@Au NP sample was entirely on account of the SiO2@Au NPs, displaying an absorbance band with a maximum peak The absorption spectra of SiO 2 showed that the absorbance band was red-shifted and broadened upon an increase in the concentration of the Au 3+ precursor, indicating the formation of larger Au NPs, which change the proximate interparticle distance (Figure 2b,c). In the absorbance band of SiO 2 @Au-NPs-treated 0 µM Au 3+ precursor, no peak was observed due to the exceedingly tiny size of the Au NPs. On the other hand, 50, 100, 150, 200, and 300 Au 3+ -treated SiO 2 @Au showed peaks at 543, 571, 593, 619, and 632 nm respectively ( Figure 2c). Moreover, the absorbance bandwidth broadened as the size of the Au NPs was increased.

Verification of the Peroxidase-like Activity of SiO 2 @Au NPs
The peroxidase-like activity of SiO 2 @Au NPs was evaluated through oxidation of a 3,3 ,5,5 -tetramethylbenzidine (TMB) substrate. The TMB oxidation reaction involves the transfer of two electrons that each produce a clear color change. When the first electron is transferred to form TMB + via oxidization of TMB, the TMB solution changes from colorless to blue. Since TMB + is quite unstable in an acidic environment, it further oxidizes to TMB 2+ when the second electron is transferred; TMB 2+ is stable in acidic conditions, exhibiting a yellow color and a maximum absorption peak at 453 nm ( Figure 3a) [63]. at 630 nm. However, a yellow solution and an absorbance band at 453 nm were observed in the TMB + H2O2 + SiO2@Au NPs sample. These results showed that SiO2@Au NPs catalyzed TMB oxidation in the presence of H2O2, indicating that a peroxidase-like reaction occurred due to the peroxidase-mimicking property of SiO2@Au NPs.

The Peroxidase-like Activity Depends on the Size of the Au NPs of the SiO2@Au NPs
To investigate the correlation between the size of the Au NPs and the peroxidase-like activity of SiO2@Au NPs, various kinds of SiO2@Au NPs with different Au NPs sizes were prepared. The concentrations of the treated Au 3+ precursors were 0, 50, 100, 150, 200, and 300 µM each, resulting in the formation of 1.4, 4.3, 6.4, 7.3, 9.5, and 15 nm diameter Au NPs on the SiO2@Au structures, respectively (Table 1 and Figure 2b). Each of these SiO2@Au NPs was subjected to the TMB assay to estimate their peroxidase-like activity. An absorbance peak at 453 nm was observed in the UV-Vis absorption spectra of all samples, indicating that all of the SiO2@Au NPs had peroxidase-like activity, irrespective of the size of the Au NPs (Supplementary Materials Figure S1). However, the SiO2@Au NPs produced without any Au 3+ precursor treatment showed relatively very weak peroxidase-like activity. This may be because the size of the Au NPs on the silica core was too small; there were a large number of vacant spaces on the SiO2@Au NPs with the given number of Au NPs, providing an insufficient To confirm the peroxidase-like activity of the SiO 2 @Au NPs, TMB + H 2 O 2 , TMB + SiO 2 @Au NPs, and TMB + H 2 O 2 + SiO 2 @Au NPs were prepared in a pH 4 buffer for the peroxidase assay. The TMB + H 2 O 2 solution was colorless, and an absorbance peak at 453 nm did not appear, as shown in Figure 3b,c. This result indicated that peroxidase-like activity did not occur in the absence of SiO 2 @Au. Next, the color of the TMB + SiO 2 @Au NP sample was entirely on account of the SiO 2 @Au NPs, displaying an absorbance band with a maximum peak at 630 nm. However, a yellow solution and an absorbance band at 453 nm were observed in the TMB + H 2 O 2 + SiO 2 @Au NPs sample. These results showed that SiO 2 @Au NPs catalyzed TMB oxidation in the presence of H 2 O 2 , indicating that a peroxidase-like reaction occurred due to the peroxidase-mimicking property of SiO 2 @Au NPs.

The Peroxidase-like Activity Depends on the Size of the Au NPs of the SiO 2 @Au NPs
To investigate the correlation between the size of the Au NPs and the peroxidase-like activity of SiO 2 @Au NPs, various kinds of SiO 2 @Au NPs with different Au NPs sizes were prepared. The concentrations of the treated Au 3+ precursors were 0, 50, 100, 150, 200, and 300 µM each, resulting in the formation of 1.4, 4.3, 6.4, 7.3, 9.5, and 15 nm diameter Au NPs on the SiO 2 @Au structures, respectively (Table 1 and Figure 2b). Each of these SiO 2 @Au NPs was subjected to the TMB assay to estimate their peroxidase-like activity. An absorbance peak at 453 nm was observed in the UV-Vis absorption spectra of all samples, indicating that all of the SiO 2 @Au NPs had peroxidase-like activity, irrespective of the size of the Au NPs (Supplementary Materials Figure S1). However, the SiO 2 @Au NPs produced without any Au 3+ precursor treatment showed relatively very weak peroxidase-like activity. This may be because the size of the Au NPs on the silica core was too small; there were a large number of vacant spaces on the SiO 2 @Au NPs with the given number of Au NPs, providing an insufficient surface area for the reaction between the Au NPs and reactants. On the other hand, SiO 2 @Au NPs treated with more than 50 µM Au 3+ precursor showed high peroxidase-like activity. The size of the Au NPs was rapidly increased when concentrations of Au 3+ precursor exceeded 50 µM, as shown in the TEM images (Figure 1b(ii). As the size of the Au NPs on the SiO 2 @Au NPs was increased, the surface area which can react with reactants also increased. Therefore, the peroxidase-like activity of the SiO 2 @Au NPs was increased as the concentrations of the Au 3+ precursor were increased (Figure 4a,b). Even though the size of the Au NPs grew as the concentration of the Au 3+ precursor increased, severe aggregation occurred immediately after the peroxidase reaction in the SiO 2 @Au was treated with over 200 µM of the Au 3+ precursor. Since good dispersibility is an important factor for generating a constant and stable catalytic activity, 150 µM of Au 3+ precursor-treated SiO 2 @Au NPs, which have high peroxidase-like activity without aggregation, was used in the subsequent experiments [64,65]. surface area for the reaction between the Au NPs and reactants. On the other hand, SiO2@Au NPs treated with more than 50 µM Au 3+ precursor showed high peroxidase-like activity. The size of the Au NPs was rapidly increased when concentrations of Au 3+ precursor exceeded 50 µM, as shown in the TEM images (Figure 1b(ii). As the size of the Au NPs on the SiO2@Au NPs was increased, the surface area which can react with reactants also increased. Therefore, the peroxidase-like activity of the SiO2@Au NPs was increased as the concentrations of the Au 3+ precursor were increased (Figure 4a,b). Even though the size of the Au NPs grew as the concentration of the Au 3+ precursor increased, severe aggregation occurred immediately after the peroxidase reaction in the SiO2@Au was treated with over 200 µM of the Au 3+ precursor. Since good dispersibility is an important factor for generating a constant and stable catalytic activity, 150 µM of Au 3+ precursor-treated SiO2@Au NPs, which have high peroxidase-like activity without aggregation, was used in the subsequent experiments [64,65].

Effects of Reaction Conditions on the Peroxidase-like Activity of SiO2@Au NPs
It is known that the catalytic activity of nanozymes is affected by reaction conditions such as those associated with an enzyme [10,32,[66][67][68][69]. For this reason, the effect of reaction conditions on the peroxidase-like activity on SiO2@Au NPs was investigated. The concentration of TMB and H2O2, pH of the buffer, the number of SiO2@Au NPs, reaction time, and termination time were considered in this study. For confirming the effects of TMB concentrations, the concentrations of TMB varied from 0 to 1.0 mM, while the other conditions were fixed in the peroxidase assay (Supplementary Materials Figure S2). The catalytic activity of SiO2@Au NPs increased until the concentrations of TMB were 0.8 mM; they then decreased at 1.0 mM, because the poor solubility of TMB in an aqueous buffer caused precipitation during the oxidation reaction (Figure 5a) [70]. To calculate the kinetic activities of SiO2@Au toward TMB concentration, various concentration of TMB in the range of 0.1 to 0.4 mM were prepared and then mixed to H2O2, and the absorbance were monitored every 10s. The absorbance at 200 s was used to calculate the Michaelis-Menten constants (Km) and the maximum reaction velocity (Vmax) in our study. The kinetic activities of SiO2@Au toward TMB concentration in the range of 0.1 to 0.4 mM TMB were plotted in Supplementary Materials Figure S3. A linear regression was found in the concentration of TMB from 0.1 to 0.4 mM. Km were obtained by using Linewaerver-Burk plots. The apparent Michaelis constant Km was calculated to be 0.060 mM and the maximum reaction velocity Vmax was 2.3 × 10 −10 M −1 •s −1 . Km of SiO2@Au was much lower than that of horseradish peroxidase enzyme, indicating that SiO2@Au has a higher affinity for TMB than horseradish peroxidase (Km = 0.438 mM). Moreo-

Effects of Reaction Conditions on the Peroxidase-like Activity of SiO 2 @Au NPs
It is known that the catalytic activity of nanozymes is affected by reaction conditions such as those associated with an enzyme [10,32,[66][67][68][69]. For this reason, the effect of reaction conditions on the peroxidase-like activity on SiO 2 @Au NPs was investigated. The concentration of TMB and H 2 O 2 , pH of the buffer, the number of SiO 2 @Au NPs, reaction time, and termination time were considered in this study. For confirming the effects of TMB concentrations, the concentrations of TMB varied from 0 to 1.0 mM, while the other conditions were fixed in the peroxidase assay (Supplementary Materials Figure S2). The catalytic activity of SiO 2 @Au NPs increased until the concentrations of TMB were 0.8 mM; they then decreased at 1.0 mM, because the poor solubility of TMB in an aqueous buffer caused precipitation during the oxidation reaction (Figure 5a) [70]. To calculate the kinetic activities of SiO 2 @Au toward TMB concentration, various concentration of TMB in the range of 0.1 to 0.4 mM were prepared and then mixed to H 2 O 2 , and the absorbance were monitored every 10s. The absorbance at 200 s was used to calculate the Michaelis-Menten constants (K m ) and the maximum reaction velocity (V max ) in our study. The kinetic activities of SiO 2 @Au toward TMB concentration in the range of 0.1 to 0.4 mM TMB were plotted in Supplementary Materials Figure S3. A linear regression was found in the concentration of TMB from 0.1 to 0.4 mM. K m were obtained by using Linewaerver-Burk plots. The apparent Michaelis constant K m was calculated to be 0.060 mM and the maximum reaction velocity V max was 2.3 × 10 −10 M −1 ·s −1 . K m of SiO 2 @Au was much lower than that of horseradish peroxidase enzyme, indicating that SiO 2 @Au has a higher affinity for TMB than horseradish peroxidase (K m = 0.438 mM). Moreover, K m of SiO 2 @Au is lower than those Au NPs (K m = 0.123 mM), glucose-oxidase-conjugated Au-attached magnetic SiO 2 microsphere (K m = 0.208 mM), Au-NPs-decorated porous silica microsphere (K m = 0.523 mM), Prussian-blue-decorated latex nanoparticle (K m = 2.19 mM), MnO 2 nanoparticles (K m = 0.083 mM), and sulfatelatex-conjugated polyelectrolyte functionalized MnO 2 NPs (K m = 0.099 mM) [71][72][73][74][75].
In the case of the concentration of hydrogen peroxide, the catalytic activity of SiO2@Au NPs was increased steeply until reaching 200 mM H2O2 and decreased at a concentration of 300 mM H2O2 (Supplementary Materials Figure S4). Even though the activity again increased at 400 mM H2O2, the rate of increase was lower than 200 mM H2O2 (Figure 5b). Subsequently, various buffers with different pH were subjected to the peroxidase assay (Supplementary Materials Figure S5). The highest activity was shown under pH 4.0, at which H2O2 was more stable and TMB dissolved maximally (Figure 5c) [10,44,70,[76][77][78]. The velocity of the catalytic reaction showed the highest value when 20 and 25 µg of SiO2@Au NPs were treated in the sample where the rest of the conditions were fixed (Figure 5d and Supplementary Materials Figure S6). About 25 min was required for the TMB + oxidation and 5 min for the termination of the TMB + oxidation to obtain stable results (Figure 5e,f).

Long-Term Stability and Reusability Test of SiO2@Au as Nanozyme
SiO2@Au NPs are substantially more advantageous over enzymes, owing to their long-term stability and reusability. Denaturation during storage and their on-off usage are the major defects when using enzymes in practice [1,2]. To verify the long-term stability of their peroxidase-like activity, the SiO2@Au NPs were examined by repeating the peroxidase assay every day, at the same time, for 14 days and on the 31st day after they were produced, keeping them under storage at 25 °C in the meantime (Figure 6a). The results showed that the peroxidase-like activity remained highly stable for at least 30 days after the fabrication of the SiO2@Au NPs. In sequence, the reusability of the SiO2@Au NPs as a nanozyme was evaluated through repeated peroxidase assays. Notably, the peroxidase-like activity of stored SiO2@Au until the fourth round was 98% of the first cycle level and mildly reduced at the fifth cycle to 89% of the first round. The reusability of SiO2@Au NPs was significantly high compared to a previous report on an Au NP-embedded silica nanostructure [79]. Moreover, SiO2@Au NPs remained 90% of catalytic activity, while SiO2@Au without the SiO2 core lost 60% of catalytic activity after five cycles of use (Supplementary Materials Figure S7). These In the case of the concentration of hydrogen peroxide, the catalytic activity of SiO 2 @Au NPs was increased steeply until reaching 200 mM H 2 O 2 and decreased at a concentration of 300 mM H 2 O 2 (Supplementary Materials Figure S4). Even though the activity again increased at 400 mM H 2 O 2 , the rate of increase was lower than 200 mM H 2 O 2 ( Figure 5b). Subsequently, various buffers with different pH were subjected to the peroxidase assay (Supplementary Materials Figure S5). The highest activity was shown under pH 4.0, at which H 2 O 2 was more stable and TMB dissolved maximally (Figure 5c) [10,44,70,[76][77][78]. The velocity of the catalytic reaction showed the highest value when 20 and 25 µg of SiO 2 @Au NPs were treated in the sample where the rest of the conditions were fixed (Figure 5d and Supplementary Materials Figure S6). About 25 min was required for the TMB + oxidation and 5 min for the termination of the TMB + oxidation to obtain stable results (Figure 5e,f).

Long-Term Stability and Reusability Test of SiO 2 @Au as Nanozyme
SiO 2 @Au NPs are substantially more advantageous over enzymes, owing to their long-term stability and reusability. Denaturation during storage and their on-off usage are the major defects when using enzymes in practice [1,2]. To verify the long-term stability of their peroxidase-like activity, the SiO 2 @Au NPs were examined by repeating the peroxidase assay every day, at the same time, for 14 days and on the 31st day after they were produced, keeping them under storage at 25 • C in the meantime (Figure 6a). The results showed that the peroxidase-like activity remained highly stable for at least 30 days after the fabrication of the SiO 2 @Au NPs. In sequence, the reusability of the SiO 2 @Au NPs as a nanozyme was evaluated through repeated peroxidase assays. Notably, the peroxidase-like activity of stored SiO 2 @Au until the fourth round was 98% of the first cycle level and mildly reduced at the fifth cycle to 89% of the first round. The reusability of SiO 2 @Au NPs was significantly high compared to a previous report on an Au NP-embedded silica nanostructure [79]. Moreover, SiO 2 @Au NPs remained 90% of catalytic activity, while SiO 2 @Au without the SiO 2 core lost 60% of catalytic activity after five cycles of use (Supplementary Materials Figure S7). These results indicate that SiO 2 @Au NPs excel not only at being highly reusable but also at separating easily from the reaction mixture. results indicate that SiO2@Au NPs excel not only at being highly reusable but also at separating easily from the reaction mixture.

Characterization
The transmission electron microscope (TEM) images of the samples were taken by using a JEM-F200 multi-purpose electron microscope (JEOL, Akishima, Tokyo, Japan) with a maximum accelerated voltage of 200 kV. The UV-Vis absorption spectra of the sample were measured by an Optizen POP UV/Vis spectrometer (Mecasys, Seoul, Korea). The centrifugation of samples was performed by using a microcentrifuge 1730R (LaboGene, Lyngen, Denmark).

Synthesis of Gold Nanoparticles (Au NPs) Assembled SiO2 Nanostructure (SiO2@Au NPs)
The SiO2@Au NPs were synthesized according to the previous report [60]. The colloidal Au was prepared by stirring 47.5 mL of water, 0.5 mL of 0.2 M NaOH, 12 µL of THPC, and 1 mL of 50 mM HAuCl4 for 1 h. Silica nanospheres (~160 nm) were prepared by using the modified Stöber method [80]. Briefly, 40 mL of EtOH, 1.6 mL TEOS, and 3 mL of NH4OH were allowed to react with each other for 20 h. The amino group was introduced to the surface of 2 mg SiO2 NPs by treating them with 62 µL of APTS. The aminated SiO2 NPs were incubated with colloidal Au (~2.5 nm) for 12 h. After several cycles of centrifugation of the mixture at 8500 rpm for 10 min, 2 mg of Au-seeded SiO2 NPs were obtained and dispersed in 2 mL of PVP solution (1 mg/mL of PVP in water). Subsequently, 200 µL of Au-seeded SiO2 NPs (1 mg/mL) suspension was added to 9.8 mL of PVP solution. Under stirring, 20 µL of 10 mM HAuCl4 solution (in water, Au 3+ precursor) and 40 µL of AA solution, (10 mM AA in water, reducing agent) were added to the mixture in sequence. The reaction mixture was stirred

Characterization
The transmission electron microscope (TEM) images of the samples were taken by using a JEM-F200 multi-purpose electron microscope (JEOL, Akishima, Tokyo, Japan) with a maximum accelerated voltage of 200 kV. The UV-Vis absorption spectra of the sample were measured by an Optizen POP UV/Vis spectrometer (Mecasys, Seoul, Korea). The centrifugation of samples was performed by using a microcentrifuge 1730R (LaboGene, Lyngen, Denmark).

Synthesis of Gold Nanoparticles (Au NPs) Assembled SiO 2 Nanostructure (SiO 2 @Au NPs)
The SiO 2 @Au NPs were synthesized according to the previous report [60]. The colloidal Au was prepared by stirring 47.5 mL of water, 0.5 mL of 0.2 M NaOH, 12 µL of THPC, and 1 mL of 50 mM HAuCl 4 for 1 h. Silica nanospheres (~160 nm) were prepared by using the modified Stöber method [80]. Briefly, 40 mL of EtOH, 1.6 mL TEOS, and 3 mL of NH 4 OH were allowed to react with each other for 20 h. The amino group was introduced to the surface of 2 mg SiO 2 NPs by treating them with 62 µL of APTS. The aminated SiO 2 NPs were incubated with colloidal Au (~2.5 nm) for 12 h. After several cycles of centrifugation of the mixture at 8500 rpm for 10 min, 2 mg of Au-seeded SiO 2 NPs were obtained and dispersed in 2 mL of PVP solution (1 mg/mL of PVP in water). Subsequently, 200 µL of Au-seeded SiO 2 NPs (1 mg/mL) suspension was added to 9.8 mL of PVP solution. Under stirring, 20 µL of 10 mM HAuCl 4 solution (in water, Au 3+ precursor) and 40 µL of AA solution, (10 mM AA in water, reducing agent) were added to the mixture in sequence. The reaction mixture was stirred for 5 min. To control the size of the Au NPs on the surface of the Au-seeded SiO 2 nanospheres, 10 mM of Au 3+ precursor and AA were added. Until the concentrations of Au 3+ reached 50, 100, 150, 200, and 300 µM in the various mixtures, the same volumes of Au 3+ precursor and AA were repeatedly added every 5 min. The SiO 2 @Au NPs were washed several times with centrifugation at 8500 rpm for 10 min. The washed SiO 2 @Au NPs were dispersed in 1 mL of 0.1% PBST solution to obtain a 0.2 mg/mL SiO 2 @Au NP suspension.

Peroxidase-like Activity of SiO 2 @Au
To verify the peroxidase-like activity of SiO 2 @Au NPs, 100 µL of TMB solution (10 mM in EtOH) and 100 µL of the various SiO 2 @Au NPs synthesized from 50, 100, 150, 200, and 300 µM Au 3+ , respectively, were added to 700 µL of pH 4 buffer. Then, freshly prepared 100 µL of H 2 O 2 solution (2 M in pH 4 buffer) was added, and the mixture was incubated for 30 min at room temperature. To terminate the reaction, 500 µL of 1 M H 2 SO 4 was added to each mixture and incubated for 10 min. The absorbance of the mixture at 350-800 nm was measured by using the UV-Vis spectrometer.

TMB Concentration
All assays of peroxidase-like activity were carried out in 1.5 mL Eppendorf tubes at room temperature. The TMB solutions were prepared in EtOH at various concentrations (1, 2, 4, 6, 8, and 10 mM, respectively). Then, 100 µL of each TMB solution, 100 µL of 2 M H 2 O 2 , and 100 µL of SiO 2 @Au (0.2 mg/mL) were added to 700 µL of pH 4 buffer. The final concentrations of TMB in the reaction mixture were 0.1, 0.2, 0.4, 0.6, 0.8, and 1.0 mM. After incubating the mixture for 30 min, 500 µL of 1 M H 2 SO 4 was added to terminate the reaction. The absorbance of mixtures was measured by using a UV-Vis spectrometer.

H 2 O 2 Concentration
Various concentrations of H 2 O 2 solutions (1, 2, 3, and 4 M) were prepared. Next, 100 µL of 6 mM TMB solution, 100 µL of each H 2 O 2 solutions, and 100 µL of SiO 2 @Au (0.2 mg/mL) were added to 700 µL of pH 4 buffer. The final concentrations of H 2 O 2 in the mixtures were 100, 200, 300, and 400 mM, respectively. After incubating the mixtures for 30 min at room temperature, we added 500 µL of 1 M H 2 SO 4 to each to terminate the reaction.

Buffer pH Value
Buffers of pH 3, 4, 5, 6, 7, and 8 were prepared. Next, 700 µL of each prepared buffer was added, followed by adding 100 µL of TMB solution, 100 µL of 2 M H 2 O 2 solution, and 100 µL of SiO 2 @Au NPs (0.2 mg/mL). The mixtures were incubated for 30 min, and the reaction was terminated by using 500 µL of 1 M H 2 SO 4 .

Reaction Time
To investigate the effect of reaction time on their peroxidase-like activity, the mixtures containing 100 µL of 6 mM TMB solution, 100 µL of SiO 2 @Au (0.2 mg/mL), and 100 µL of 2 M H 2 O 2 solution were added to 700 µL of pH 4 buffer. Next, the mixtures were incubated for 0, 5, 10, 15, 20, 25, and 30 min respectively and terminated by using 1 M H 2 SO 4 .

Long-Term Stability of Peroxidase-like Activity
To investigate the long-term stability of peroxidase-like activity of SiO 2 @Au, the experiment was repeated every day for 2 weeks and on the 31st day after their fabrication. The experimental procedures were conducted as follows: adding 100 µL of 6 M TMB solution, 100 µL of SiO 2 @Au (0.2 mg/mL), and 100 µL of 2 M H 2 O 2 solution to 700 µL of pH 4 buffer; 30 min reaction time; 10 min for termination, using 500 µL of 1 M H 2 SO 4 .

Reusability as Nanozymes
To investigate the reusability of SiO 2 @Au, the peroxidase assay was performed according to the mentioned procedures. After the assay was performed, the SiO 2 @Au NPs were collected by using centrifugation at 10,000 rpm for 10 min, and the absorbance of the supernatant at 453 nm was measured by using a UV-Vis spectrometer. The peroxidase assay was repeated with the collected SiO 2 @Au NPs.

Conclusions
In summary, we successfully synthesized finely controllably sized Au NPs on the SiO 2 nanosphere (SiO 2 @Au), using the seed-mediated growth method and interval dropping method under mild conditions. The effect of the size of Au NPs on the SiO 2 @Au NPs was confirmed by the TEM images, color changing of its suspension, and UV-Vis absorption spectra. Moreover, we investigated the factors affecting the peroxidase-like activity of SiO 2 @Au NPs, such as TMB concentration, H 2 O 2 concentration, pH, SiO 2 @Au NPs amount, reaction time, and termination time. Furthermore, SiO 2 @Au NPs showed high stability during the 30-day-long storage time at room temperature and outstanding reusability for five cycles. This work is therefore meaningful for utilizing controllable nanoparticles in various fields and provides a better approach to develop nanoparticle-based nanozymes.