Synthesis of Gold-Platinum Core-Shell Nanoparticles Assembled on a Silica Template and Their Peroxidase Nanozyme Properties

Bimetallic nanoparticles are important materials for synthesizing multifunctional nanozymes. A technique for preparing gold-platinum nanoparticles (NPs) on a silica core template (SiO2@Au@Pt) using seed-mediated growth is reported in this study. The SiO2@Au@Pt exhibits peroxidase-like nanozyme activity has several advantages over gold assembled silica core templates (SiO2@Au@Au), such as stability and catalytic performance. The maximum reaction velocity (Vmax) and the Michaelis–Menten constants (Km) were and 2.1 × 10−10 M−1∙s−1 and 417 µM, respectively. Factors affecting the peroxidase activity, including the quantity of NPs, solution pH, reaction time, and concentration of tetramethyl benzidine, are also investigated in this study. The optimization of SiO2@Au@Pt NPs for H2O2 detection obtained in 0.5 mM TMB; using 5 µg SiO2@Au@Pt, at pH 4.0 for 15 min incubation. H2O2 can be detected in the dynamic liner range of 1.0 to 100 mM with the detection limit of 1.0 mM. This study presents a novel method for controlling the properties of bimetallic NPs assembled on a silica template and increases the understanding of the activity and potential applications of highly efficient multifunctional NP-based nanozymes.

Several studies have been conducted on bimetallic NPs owing to the increased awareness of the options to adjust their physical and chemical properties. Compared with

Preparation of Au@Pt NPs-Assembled Silica Nanostructures
The seed-mediated growth from SiO 2 @Au seeds was used to prepare the gold-platinumembedded silica nanospheres (SiO 2 @Au@Pt). First, the surface SiO 2 template was assembled using small Au NPs (2.6 ± 0.52 nm) (Figure 1a). Then, 10 mM H 2 PtCl 4 and AA solutions were added dropwise into the dispersion of SiO 2 @Au seeds to obtain a final Pt 2+ concentration of 200 µM. The Au@Pt NPs on the surface of the SiO 2 core were bigger (3.6 ± 0.56 nm) than SiO 2 @Au seeds. The presence of Pt on the surface of SiO 2 @Au was confirmed by the line energy dispersive X-ray (EDS) mapping of SiO 2 @Au@Pt (Figures 1b  and S1). In Figure 1b, the signal of both Pt and Au elements could obtain in the image. The quantitative EDS analysis in Table S1 of SiO 2 @Au@Pt synthesized at 200 µM Pt 2+ consists of 74.6% Pt and 23.6% Au as mentioned in Table S1. of synthesis, SiO2@Au@Pt was used as a peroxidase-like nanomaterial to detect H2O2 efficiently.

Preparation of Au@Pt NPs-Assembled Silica Nanostructures
The seed-mediated growth from SiO2@Au seeds was used to prepare the goldplatinum-embedded silica nanospheres (SiO2@Au@Pt). First, the surface SiO2 template was assembled using small Au NPs (2.6 ± 0.52 nm) (Figure 1a). Then, 10 mM H2PtCl4 and AA solutions were added dropwise into the dispersion of SiO2@Au seeds to obtain a final Pt 2+ concentration of 200 µM. The Au@Pt NPs on the surface of the SiO2 core were bigger (3.6 ± 0.56 nm) than SiO2@Au seeds. The presence of Pt on the surface of SiO2@Au was confirmed by the line energy dispersive X-ray (EDS) mapping of SiO2@Au@Pt (Figures 1b  and S1). In Figure 1b, the signal of both Pt and Au elements could obtain in the image. The quantitative EDS analysis in Table S1 of SiO2@Au@Pt synthesized at 200 µM Pt 2+ consists of 74.6% Pt and 23.6% Au as mentioned in Table S1. The UV-Vis spectroscopy of SiO2@Au@Pt was conducted when the Pt 2+ solution was added into the SiO2@Au seed suspension in the presence of AA. The absorbance intensity of SiO2@Au showed a slight increase at ~500 nm. This peak was suppressed in the absorbance spectrum of SiO2@Au@Pt but the spectrum of SiO2@Au@Pt increased in the UV region (Figure 1c) owing to the Pt layer, consistent with previous results of pure Pt hydrosol [66,67]. The results of TEM, EDS mapping, UV-Vis spectrum of SiO2@Au@Pt indicated that Pt was deposited to the SiO2@Au.

Peroxidase-like Activity of SiO2@Au@Pt NPs
The peroxidase-like activity of SiO2@Au@Pt NPs was evaluated using the oxidation reaction of 3,3′,5,5′-tetramethylbenzidine (TMB) substrate prepared in a buffer (pH = 4) The UV-Vis spectroscopy of SiO 2 @Au@Pt was conducted when the Pt 2+ solution was added into the SiO 2 @Au seed suspension in the presence of AA. The absorbance intensity of SiO 2 @Au showed a slight increase at~500 nm. This peak was suppressed in the absorbance spectrum of SiO 2 @Au@Pt but the spectrum of SiO 2 @Au@Pt increased in the UV region ( Figure 1c) owing to the Pt layer, consistent with previous results of pure Pt hydrosol [66,67]. The results of TEM, EDS mapping, UV-Vis spectrum of SiO 2 @Au@Pt indicated that Pt was deposited to the SiO 2 @Au.

Peroxidase-like Activity of SiO 2 @Au@Pt NPs
The peroxidase-like activity of SiO 2 @Au@Pt NPs was evaluated using the oxidation reaction of 3,3 ,5,5 -tetramethylbenzidine (TMB) substrate prepared in a buffer (pH = 4) containing TMB or a TMB-H 2 O 2 mixture. TMB oxidation involves the exchange of two electrons. When TMB transfers one electron to form TMB + , the solution changes from colorless to blue. However, TMB + is unstable in acidic conditions and must oxidize to TMB 2+ , forming a yellow solution [68]. In this study, the TMB, H 2 O 2 , and TMB+H 2 O 2 solutions without SiO 2 @Au@Pt NPs were colorless and no absorbance peaks were observed at 453 nm (Figure 1d), indicating that there was no peroxidase-like catalytic activity in the absence of SiO 2 @Au@Pt. A small absorbance band was observed for the SiO 2 @Au@Pt + TMB solution, and a strong absorbance band with peaks at 370 and 652 nm was observed for the SiO 2 @Au@Pt + TMB-H 2 O 2 solution. The SiO 2 @Au@Pt + TMB-H 2 O 2 solution changed from blue to yellow and an absorbance band with a peak at 453 nm was observed ( Figure  S2a). These results indicate that the conversion of TMB to TMB 2+ was catalyzed by the SiO 2 @Au@Pt NPs with H 2 O 2 , suggesting that SiO 2 @Au@Pt NPs has a peroxidase-like activity. To confirm the synergic qualities of SiO 2 @Au@Pt, an Au layer was deposited on the surface of the SiO 2 @Au seeds using a 200 µM Au 3+ solution. The peroxidase-like catalytic activity of SiO 2 @Au@Pt (5 µg) and SiO 2 @Au@Au (5 µg containing TMB or a TMB-H2O2 mixture. TMB oxidation involves the exchange of tw electrons. When TMB transfers one electron to form TMB + , the solution changes from colorless to blue. However, TMB + is unstable in acidic conditions and must oxidize t TMB 2+ , forming a yellow solution [68]. In this study, the TMB, H2O2, and TMB+H2O solutions without SiO2@Au@Pt NPs were colorless and no absorbance peaks wer observed at 453 nm (Figure 1d), indicating that there was no peroxidase-like catalyti activity in the absence of SiO2@Au@Pt. A small absorbance band was observed for th SiO2@Au@Pt + TMB solution, and a strong absorbance band with peaks at 370 and 652 nm was observed for the SiO2@Au@Pt + TMB-H2O2 solution. The SiO2@Au@Pt + TMB-H2O solution changed from blue to yellow and an absorbance band with a peak at 453 nm wa observed ( Figure S2a). These results indicate that the conversion of TMB to TMB 2+ wa catalyzed by the SiO2@Au@Pt NPs with H2O2, suggesting that SiO2@Au@Pt NPs has peroxidase-like activity. To confirm the synergic qualities of SiO2@Au@Pt, an Au laye was deposited on the surface of the SiO2@Au seeds using a 200 µM Au 3+ solution. Th peroxidase-like catalytic activity of SiO2@Au@Pt (5 µg) and SiO2@Au@Au (5 µg  The blue color of the SiO2@Au@Pt in TMB-H2O2 is darker and the absorbanc intensity at 370 and 652 nm is stronger than those of the SiO2@Au@Au suspension. Th absorbance intensities of the SiO2@Au@Pt suspension were 5.7-and 7.7-fold of those o SiO2@Au@Au at 370 and 652 nm, respectively. Similarly, the absorbance intensity of th SiO2@Au@Pt suspension at 453 nm was stronger than that of SiO2@Au@Au ( Figure S3b In addition, the recycling of both SiO2@Au@Au and SiO2@Au@Pt in the TMB-H2O solution at 453 nm was compared when the NPs were reused five times. The absorbanc intensity of SiO2@Au@Au in a TMB-H2O2 solution at 453 nm decreased to approximatel The blue color of the SiO 2 @Au@Pt in TMB-H 2 O 2 is darker and the absorbance intensity at 370 and 652 nm is stronger than those of the SiO 2 @Au@Au suspension. The absorbance intensities of the SiO 2 @Au@Pt suspension were 5.7-and 7.7-fold of those of SiO 2 @Au@Au at 370 and 652 nm, respectively. Similarly, the absorbance intensity of the SiO 2 @Au@Pt suspension at 453 nm was stronger than that of SiO 2 @Au@Au ( Figure S3b). In addition, the recycling of both SiO 2 @Au@Au and SiO 2 @Au@Pt in the TMB-H 2 O 2 solution at 453 nm was compared when the NPs were reused five times. The absorbance intensity of SiO 2 @Au@Au in a TMB-H 2 O 2 solution at 453 nm decreased to approximately 40% while that of SiO 2 @Au@Pt in a TMB-H 2 O 2 solution at 453 nm decreased to 80%. These results indicate that the catalytic ability of SiO 2 @Au@Pt is better than that of SiO 2 @Au@Au, and that SiO 2 @Au@Pt NPs are reusable and separatable from the reaction mixture.
The catalytic performance of SiO 2 @Au@Pt NPs at TMB concentrations of 0-600 µM was also investigated as the absorbance intensity at 652 nm in TMB-H 2 O 2 every 3 min (Figure 2b). The absorbance intensity of the oxidation of TMB increased as the TMB concentration increased, following Michaelis-Menten behavior. The signal of SiO 2 @Au@Pt NPs increased as the TMB concentration increased from 100 to 500 µM and reached saturation at 600 µM. The relationship of the TMB concentration and absorbance intensity at 652 nm after incubation for 180 s was plotted according to the Lineweaver-Burk equation to calculate the maximum reaction velocity (V max ) and the Michaelis-Menten constants (K m ) ( Figure 2d). The kinetic activity of SiO 2 @Au@Pt at various TMB concentrations (100-500 µM) revealed a linear relationship. The K m was 417 µM and the V max was 2.1 × 10 −10 M −1 ·s −1 . The K m of SiO 2 @Au@Pt indicates a higher affinity of SiO 2 @Au@Pt for TMB compared with that for horseradish peroxidase enzyme (K m = 438 µM). The K m of SiO 2 @Au@Pt was higher than those of Au NPs (K m = 123 µM), SiO 2 @Au@Au NPs (Km = 60 µM), glucose oxidase-conjugated Au-attached SiO 2 microspheres (K m = 208 µM), MnO 2 NPs (K m = 83 µM), and latex-conjugated MnO 2 NPs (K m = 99 µM). The K m of SiO 2 @Au@Pt was lower than those of Au NPs-decorated porous silica microspheres (K m = 523 µM) and Prussianblue-decorated latex NPs (K m = 2.19 mM) [55]. The V max of SiO 2 @Au@Pt was higher than that of SiO 2 @Au@Au (V max = 2.1 × 10 −10 M −1 ·s −1 ), indicating that SiO 2 @Au@Pt oxidizes TMB at a faster rate than SiO 2 @Au@Au. SiO 2 @Au@Pt had a comparatively more stable catalytic activity that remained at 80% after five reuses (Figure 2b).

Effects of Synthesis and Experimental Conditions on the Catalytic Activity of SiO 2 @Au@Pt NPs
Various concentrations of the Pt 2+ precursor (100-400 µM) were added to the SiO 2 @Au seeds. The size of Pt increased as the Pt 2+ concentration increased (Figures 3a and S2). In particularly, the size of Au@Pt synthesized at 100, 200, 300, 400 µM Pt 2+ were 3.1 ± 0.61; 3.6 ± 0.56; 3.9 ± 0.59; 4.6 ± 0.62 nm, respectively. At a high concentration of Pt 2+ (>200 µM), the Au@Pt on the SiO 2 surface partly merged. The quantitative EDS analysis of Pt and Au elements on the surface of SiO 2 synthesized at various Pt 2+ concentration was carried out to investigate the composition of the Au@Pt on the SiO 2 surface and the results were shown in Table S1. All Au@Pt NPs contained both Pt and Au elements, but their Au and Pt components were different. The atomic Pt component increased from 71.59% to 87.07% while the atomic Au component decreased from 28.41 to 12.93% when Pt 2+ increased from 100 µM to 400 µM. Therefore, the reciprocal of Pt and Au increased from 2.5 (at 100 µM Pt 2+ ) to 6.7 (at 400 µM Pt 2+ ). The results matched to the TEM images, and it indicated that Pt was gradually deposited on the surface of SiO 2 @Au. The absorption spectra of SiO2@Au@Pt were collected (Figure 3b). The absorbance intensity at 300-800 nm increased as the concentration of the Pt 2+ precursor increased, indicating the formation of larger Au@Pt NPs ( Figure S2) that subsequently affected the proximate interparticle distance. The absorbance of the suspension broadened as the size of the Au@Pt NPs increased ( Figure S2). The growth of Au@Pt NPs on SiO2 could be controlled well (Figure 3).
The correlation between the peroxidase-like activity of SiO2@Au@Pt NPs and the concentration of Pt 2+ was investigated (Figure 3c). The peroxidase-like activity of SiO2@Au@Pt NPs synthesized with 100-400 µM Pt 2+ was estimated using a TMB assay. The SiO2@Au seeds showed very weak peroxidase-like activity because of the lack of spaces on the SiO2@Au NPs and the small Au NPs on the SiO2 template, resulting an insufficient surface area for the catalytic reaction between the Au NPs and the TMB-H2O2 mixture [55]. The UV-Vis absorption spectra of all SiO2@Au@Pt NPs showed an absorbance peak at 453 nm (Figure 3c-e), indicating that all of the SiO2@Au@Pt NPs had peroxidase-like activity that was dependent on the initial Pt 2+ concentration.
In contrast, SiO2@Au@Pt NPs treated with a Pt 3+ precursor concentration >100 µM had high peroxidase-like activity. As the concentration of Pt 2+ increased, the size of the Au@Pt NPs on the SiO2@Au@Pt NPs increased from 3.1 to 4.6 nm, which increased the surface area for reactions between NPs and reactants. Therefore, the catalytic activity of SiO2@Au@Pt NPs increased as the concentration of the Pt 2+ precursor increased from 100 to 200 µM because of the formation of sublayers or a monolayer of Pt on the surface of SiO2@Pt [45,67]. This result consistent with the previous report where the increase of Pt component of Au-Pt led an better catalytic activity because the alloying of Pt with Au can change the electronic structure of Pt, leading the catalytic performance of Au@Pt changes [11,37,69]. Although the Au@Pt NPs grew as the concentration of the Pt 2+ precursor increased, the peroxidase reaction of SiO2@Au@Pt treated with >200 µM of the Pt 2+ precursor did not increase because TMB and H2O2 cannot gain access to inner part of the thick Pt layer [66]. It means that the catalytic activities of SiO2@Au@Pt reached the highest value at the Pt/Au ratio of 3.2 and decreased when Pt 2+ concentration increased further. The absorption spectra of SiO 2 @Au@Pt were collected (Figure 3b). The absorbance intensity at 300-800 nm increased as the concentration of the Pt 2+ precursor increased, indicating the formation of larger Au@Pt NPs ( Figure S2) that subsequently affected the proximate interparticle distance. The absorbance of the suspension broadened as the size of the Au@Pt NPs increased ( Figure S2). The growth of Au@Pt NPs on SiO 2 could be controlled well (Figure 3).
The correlation between the peroxidase-like activity of SiO 2 @Au@Pt NPs and the concentration of Pt 2+ was investigated (Figure 3c). The peroxidase-like activity of SiO 2 @Au@Pt NPs synthesized with 100-400 µM Pt 2+ was estimated using a TMB assay. The SiO 2 @Au seeds showed very weak peroxidase-like activity because of the lack of spaces on the SiO 2 @Au NPs and the small Au NPs on the SiO 2 template, resulting an insufficient surface area for the catalytic reaction between the Au NPs and the TMB-H 2 O 2 mixture [55]. The UV-Vis absorption spectra of all SiO 2 @Au@Pt NPs showed an absorbance peak at 453 nm (Figure 3c-e), indicating that all of the SiO 2 @Au@Pt NPs had peroxidase-like activity that was dependent on the initial Pt 2+ concentration.
In contrast, SiO 2 @Au@Pt NPs treated with a Pt 3+ precursor concentration >100 µM had high peroxidase-like activity. As the concentration of Pt 2+ increased, the size of the Au@Pt NPs on the SiO 2 @Au@Pt NPs increased from 3.1 to 4.6 nm, which increased the surface area for reactions between NPs and reactants. Therefore, the catalytic activity of SiO 2 @Au@Pt NPs increased as the concentration of the Pt 2+ precursor increased from 100 to 200 µM because of the formation of sublayers or a monolayer of Pt on the surface of SiO 2 @Pt [45,67]. This result consistent with the previous report where the increase of Pt component of Au-Pt led an better catalytic activity because the alloying of Pt with Au can change the electronic structure of Pt, leading the catalytic performance of Au@Pt changes [11,37,69]. Although the Au@Pt NPs grew as the concentration of the Pt 2+ precursor increased, the peroxidase reaction of SiO 2 @Au@Pt treated with >200 µM of the Pt 2+ precursor did not increase because TMB and H 2 O 2 cannot gain access to inner part of the thick Pt layer [66]. It means that the catalytic activities of SiO 2 @Au@Pt reached the highest value at the Pt/Au ratio of 3.2 and decreased when Pt 2+ concentration increased further. Therefore, 200 µM of Pt 2+ precursor-treated SiO 2 @Au@Pt NPs, which exhibited high peroxidase-like activity, were used in subsequent experiments.
Reaction conditions affect the catalytic activity of nanozymes similarly to the effects of reaction conditions on enzymes [21,[70][71][72][73][74]. Therefore, the peroxidase-like activity of different amounts of SiO 2 @Au@Pt NPs were investigated at different incubation times, pH values of the buffer solution, and TMB concentrations (Figure 4).

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Reaction conditions affect the catalytic activity of nanozymes similarly to the effects of reaction conditions on enzymes [21,[70][71][72][73][74]. Therefore, the peroxidase-like activity of different amounts of SiO2@Au@Pt NPs were investigated at different incubation times, pH values of the buffer solution, and TMB concentrations (Figure 4). The amount of SiO2@Au@Pt varied from 0.02 to 20 µg (Figures 4a and S3). The absorbance intensity of SiO2@Au@Pt in a TMB-H2O2 solution at 453 nm increased as the amount of SiO2@Au@Pt increased from 0.02 to 5 µg. When the amount of SiO2@Au@Pt increased to >10 µg, the poor solubility of TMB in the aqueous solution resulted in significant aggregation, inducing precipitation with oxidation [55].
The absorbance intensity increased as the incubation time increased from 5 to 15 min (Figures 4b and S4). The absorbance intensity reached saturation after 15 min of incubation.
The highest peroxidase catalytic activity was obtained at a pH of 4.0, at which TMB dissolved maximally and H2O2 was the most stable (Figures 4c and S5), which is consistent with the results of previous studies [17,21,[75][76][77][78].
The absorbance of TMB 2+ at 453 nm increased as the TMB concentration increased and reached the saturation at 600 µM TMB (Figures 4d and S6).

Effects of H2O2 Concentration on Peroxidase-like Activity of SiO2@Au@Pt NPs
After optimizing the detection conditions, the absorbance intensity was measured at 0-400 nm for the SiO2@Au@Pt in 500 µM TMB with various concentrations of H2O2 ( Figure  5). The yellow color of the 5 µg SiO2@Au@Pt suspension became darker as the H2O2 concentration increased to 200 mM, indicating that more TMB 2+ was produced as the H2O2 The amount of SiO 2 @Au@Pt varied from 0.02 to 20 µg (Figures 4a and S3). The absorbance intensity of SiO 2 @Au@Pt in a TMB-H 2 O 2 solution at 453 nm increased as the amount of SiO 2 @Au@Pt increased from 0.02 to 5 µg. When the amount of SiO 2 @Au@Pt increased to >10 µg, the poor solubility of TMB in the aqueous solution resulted in significant aggregation, inducing precipitation with oxidation [55].
The absorbance intensity increased as the incubation time increased from 5 to 15 min (Figures 4b and S4). The absorbance intensity reached saturation after 15 min of incubation.
The highest peroxidase catalytic activity was obtained at a pH of 4.0, at which TMB dissolved maximally and H 2 O 2 was the most stable (Figures 4c and S5), which is consistent with the results of previous studies [17,21,[75][76][77][78].
The absorbance of TMB 2+ at 453 nm increased as the TMB concentration increased and reached the saturation at 600 µM TMB (Figures 4d and S6).

Effects of H 2 O 2 Concentration on Peroxidase-like Activity of SiO 2 @Au@Pt NPs
After optimizing the detection conditions, the absorbance intensity was measured at 0-400 nm for the SiO 2 @Au@Pt in 500 µM TMB with various concentrations of H 2 O 2 ( Figure 5). The yellow color of the 5 µg SiO 2 @Au@Pt suspension became darker as the H 2 O 2 concentration increased to 200 mM, indicating that more TMB 2+ was produced as the H 2 O 2 concentration increased. The catalytic activity of SiO 2 @Au@Pt increased as the H 2 O 2 concentration increased to 200 mM and reached saturation at 300 mM because of aggregation ( Figure S7).
A linear curve-fitting procedure was used to calibrate the reaction (Figure 5c). A significant relationship was found between the absorbance intensity at 453 nm and the H 2 O 2 concentration from 1.0 to 100 mM (calibration curve: y = 0.0185 x + 0.63285, where x is the H 2 O 2 concentration, y is the absorbance intensity at 453 nm, and R 2 = 0.99). The theoretical LOD was 1.0 mM, estimated using the 3sblank criterion. This LOD is higher than the LOD of silver NPs modified cellulose nanowhiskers [79], polyoxometalate [80], Ce 2 (WO 4 ) 3 , papain [81], Ag-nanoparticle-decorated silica microspheres [82] and magnetic mesoporous silica nanoparticles [83]. These results suggest that this material can be used to detect H 2 O 2 .

Characterization
The TEM images of the samples were obtained using a JEM-F200 electron microscope (JEOL, Akishima, Tokyo, Japan) at an accelerated voltage of 200 kV. The UV-Vis spectra of the samples were recorded using an Optizen POP UV/Vis spectrometer (Mecasys, Seoul, Korea). The samples were centrifuged using a 1730R microcentrifuge (LaboGene,

Characterization
The TEM images of the samples were obtained using a JEM-F200 electron microscope (JEOL, Akishima, Tokyo, Japan) at an accelerated voltage of 200 kV. The UV-Vis spectra of the samples were recorded using an Optizen POP UV/Vis spectrometer (Mecasys, Seoul, Korea). The samples were centrifuged using a 1730R microcentrifuge (LaboGene, Lyngen, Denmark).

Synthesis of Gold-Platinum Nanoparticles
Assembled on a SiO 2 Nanostructure (SiO 2 @Au@Pt NPs) The SiO 2 @Au seed NPs were synthesized as previously reported [54]. Briefly, colloidal Au NPs were prepared from HAuCl 4 and THPC. Silica templates (approximately 160 nm) were prepared using the modified Stöber method [84]. The surfaces of 50 mg SiO 2 NPs were modified using amino groups via incubation with 62 µL APTS overnight at 25 • C. Animated SiO 2 NPs (2 mg) were incubated with 10 mL colloidal Au (approximately 2.5 nm) for 12 h at 25 • C. After the suspension was centrifuged for 10 min at 8500 rpm and washed with EtOH, 2 mg of SiO 2 @Au seed NPs were dispersed in 2 mL of 1 mg/mL PVP solution. Subsequently, 200 µL of SiO 2 @Au seed (1 mg/mL) suspension was mixed with 9.8 mL of PVP solution. Under stirring, 20 µL of 10 mM HPtCl 6 solution (in water, Pt 2+ precursor) and 40 µL of AA reducing agent (10 mM AA in water) were added to the mixture. The mixture was reacted for 5 min under stirring to convert Pt 2+ to Pt(0). The same volumes of Pt 2+ precursor and AA were added every 5 min to obtain the desired concentration of Pt 2+ . The SiO 2 @Au@Pt NPs were then carefully washed with EtOH several times using centrifugation at 8500 rpm for 10 min. The washed SiO 2 @Au@Pt NPs were redispersed in 0.1% PBST solution (1 mL) to obtain a 0.2 mg/mL SiO 2 @Au@Pt NP suspension.

Peroxidase-like Activity of SiO 2 @Au@Pt NPs
To verify the peroxidase-like catalytic activity of SiO 2 @Au@Pt NPs, TMB solution (6 mM in EtOH, 100 µL), the SiO 2 @Au@Pt NPs suspension (100 µL) synthesized with 100, 200, 300, and 400 µM Pt 2+ , and freshly prepared H 2 O 2 solution (2 M in pH 4 buffer, 100 µL) were added to 700 µL of buffer (pH = 4.0). Then, the mixture was incubated at 25 • C for 15 min. To terminate the reaction, 1 M H 2 SO 4 solution (500 µL) was added to each mixture and the resulting mixture was incubated at 25 • C for 10 min. The absorbances of the suspensions were measured at 300-1,000 nm using a UV-Vis spectrometer.

PH Value of the Buffer
A mixture containing 6 mM TMB (100 µL), a SiO 2 @Au@Pt NP suspension (0.05 mg/mL, 100 µL), and 2 M H 2 O 2 (100 µL) was added to 700 µL of buffer at a pH range from 3.0 to 11.0. The mixture was incubated for 15 min and terminated using 1 M H 2 SO 4 (500 µL). The absorbances of the mixtures were measured at 453 nm using a UV-Vis spectrometer.

Conclusions
In summary, SiO 2 @Au@Pt NPs were successfully synthesized using the seed-mediated growth method under mild conditions. Compared with SiO 2 @Au@Au, the SiO 2 @Au@Pt NPs exhibited more synergic and stable catalytic abilities that remained at 80% after five uses of 5 µg SiO 2 @Au@Pt NPs. In addition, the peroxidase-like activity of SiO 2 @Au@Pt NPs under various conditions such as the amount of SiO 2 @Au@Pt NPs, pH of the buffer solution, incubation time, and TMB concentration were also investigated, revealing optimized conditions of 5 µg SiO 2 @Au@Pt at pH 4.0, with 15 min of incubation in the presence of 500 µM TMB. SiO 2 @Au@Pt was used to detect H 2 O 2 . The dynamic linear range was obtained from 1 to 100 mM, with an LOD of 1.0 mM. Therefore, this study suggests novel uses of bimetallic metal-assembled silica nanostructures in various fields and provides a suitable method for the development of nanoparticle-based multi-functional nanozymes.