The synthesis steps are as follows: SA (0.1 g) was added into 50 mL of 1% (v/v) acetic acid solution and completely stirred for about 15 min. Then, 2 mL H₂PtCl₆ (10 mM) was mixed with 47 mL SA solution. The mixture was vortex at ambient temperature for 30 min. One milliliters of newly prepared NaBH₄ solution (70 mM) was added to the mixture and was completed within 5 min. The SA-PtNPs (78.03 mg/L) were obtained by stirring in the dark for 90 min. The SA-PtNPs was transferred in a dialysis bag and dialysed against water. The dialysis water was replaced after 1 h followed by frequent changes for 48 h. Finally, the SA-PtNPs were preserved constantly in darkness at 4°C.

Fifty μL of various concentrations of OPC, 50 μL of TMB (3 mM), and 30 μL of SA-PtNPs (15.61 mg/L) were added into 870 μL of phosphate buffer (pH = 4.5, 50 mM). The mixture was then incubated for 5 min at 37°C. Subsequently, 200 μL of H₂SO₄ (2 M) was introduced to the reaction as a stop buffer. Finally, the solution was transferred to a quartz cell for measurement at 450 nm. Other experimental part can be seen in Supplementary Material.

SA-PtNPs were prepared by a simple approach with SA as a stabilizing agent and NaBH₄ as a reductant. The stabilizing and reducing agents have pivotal effects during the synthesis of SA-PtNPs, thus the concentrations of SA and NaBH₄ had to be determined, respectively (Supplementary Figure 1). As shown in Supplementary Figure 1, the concentrations of SA and NaBH₄ on the preparation of SA-PtNPs were optimized to be 2 mg/mL and 1.4 mM. The images in Figure 1A display a yellowish-brown product and a state of well-dispersion. Moreover, the high-resolution transmission electron microscope (TEM) image shows that the crystal plane spacing of the SA-PtNPs is 0.19 nm. The size distribution of SA-PtNPs determined from 100 random nanoparticles is shown in Supplementary Figure 2A with an average diameter of 5.9 ± 0.6 nm. The energy dispersive X-ray spectroscopy (EDS) characterization results of SA-PtNPs (Supplementary Figure 2B) indicate that Pt has a peak at the corresponding elemental feature, indicating the presence of Pt in the material. The infrared spectra of SA and SA-PtNPs are shown in Figure 1B. It can be seen that the stretching vibration band of O-H at 3,471 cm⁻¹, the stretching vibration band of C-H at 2,932 cm⁻¹, and the glycoside skeleton absorption peaks of C-O-C at 1,030 and 810 cm⁻¹ are invariable. However, the peaks suggesting -COO- in the molecule at 1,613 and 1,412 cm⁻¹ shift to higher wavenumbers of 1,634 cm⁻¹ and 1,448 cm⁻¹, respectively. These changes probably suggested that the -COO- groups of SA are responsible for interacting with PtNPs.

As shown in Figure 1C, X-ray diffraction (XRD) characterization of SA-PtNPs at 39.6°, 46.1°, 67.2°, and 81.2°can be indexed well to the diffraction from (111), (200), (220), and (311) planes of face-centered cubic (fcc) Pt (JCPDS 04-0802), respectively, proving that Pt exists in the crystalline form. Furthermore, the surface elemental analysis of SA-PtNPs was also performed by X-ray photoelectron spectroscopy (XPS). The characteristic spectrum can be mainly decomposed into two pairs of dual states of Pt 4f_7/2 and Pt 4f_5/2. These peaks with the intense distinct peak are concentrated in the binding energies around 71.57 and 75.02 eV, which could be attributed to the existence of metallic platinum (Pt⁰). The other peaks at around 75.47 and 78 eV were contributed to the Pt⁴⁺ (Cho and Ouyang, 2011; Wu et al., 2014), and the relative contents of Pt⁰ and Pt⁴⁺ are 42.7 and 57.3% (Figure 1D).

To evaluate the oxidase-like activity of SA-PtNPs, TMB was chosen as the chromogenic substrate in this study. Figure 2A shows that SA-PtNPs can quickly catalyze the oxidation of TMB under O₂ to generate a blue outcome with peaks absorption at 370 and 652 nm without of H₂O₂. Since the absorbance at 450 nm after the addition of H₂SO₄ can be stable for a long time with a complete spectrum shift and concomitant hyperchromic effect (Bos et al., 1981; Frey et al., 2000). Therefore, the oxidization reaction in this work was stopped by H₂SO₄, resulting in a yellow substance generated with a maximum absorbance at 450 nm. It's worth mentioning that the oxidase-like activity of SA is so low that their effect on SA-PtNPs can be ignored (Supplementary Figure 3).

Some other chromogenic substrates were also tested to demonstrate the versatility of SA-PtNPs, including o-phenylenediamine (OPD), pyrogallol, 2,2′-azino-bis(3-ethylben- zothiazoline-6-sulfonic acid) diammonium salt (ABTS), 4-aminoantipyrine (4-AAP) and N-ethyl-N-(3-sulfopropyl)-3-methylaniline sodium salt (TOPS) (Supplementary Figure 4). Thus, these results verified that SA-PtNPs can act as a new type of oxidase mimic. For further application of the SA-PtNPs-TMB system, pH, temperature, and TMB concentration were optimized to be 4.5, 37°C, and 0.15 mM, respectively (Supplementary Figure 5).

The steady-state dynamics assay of the SA-PtNPs-TMB system is depicted in Figure 2B with the reaction rate (v) depending on TMB concentration. With the increase of TMB concentration, reaction rate increased quickly at first and then changed slowly. To further study the kinetics, the Michaelis Constant (K_m) value of SA-PtNPs was counted to be 11.6 μM fitting by Michaelis-Menten equation. The K_m is an important parameter used to evaluate the affinity of nanozyme to substrate and to discuss the nanozyme-substrate interaction. Table 1 also shows the comparison of the K_m value of oxidase mimics and points out that SA-PtNPs have a remarkably high affinity for TMB. As shown in Supplementary Figure 6, the specific activity value of SA-PtNP has been evaluated as 2,711 U/g according to the fitted straight line (Supplementary Figure 6B) (Jiang et al., 2018).

To determine the mechanism, possible superoxide anions (O2•-) were also studied. First of all, SA-PtNPs-catalyzed TMB oxidation was conducted in air with dissolved oxygen and bubbled with highly pure nitrogen gas, respectively. The reaction rate of SA-PtNPs-catalyzed TMB oxidation was found to decrease rapidly under nitrogen atmosphere, which indicates that dissolved O₂ is involved in the process (Supplementary Figure 7). Since ascorbic acid (AA) could eliminate O2•- (Cai et al., 2018), we performed AA oxidations. As Figure 2C shows that AA has an absorption peak at ~250 nm in the wavelength range. A slight variation at 250 nm and oxidation of AA occurred while SA-PtNPs or O₂ was removed. However, when SA-PtNPs and O₂ co-exist, AA was consumed. In this phenomenon, the presence of O2•- can be ruled out in the SA-PtNPs-TMB system. Furthermore, the oxygen reduction reaction (ORR) assay was carried out to further verify the mechanism. Relevant experiments were carried out with using carbon electrode as electrochemical working electrode which was modified with SA-PtNPs. No peak appear after the mixed solution is filled with nitrogen (Figure 2D). It produced a cathode reduction peak at ~-0.34 V in an environment with oxygen. On the basis of these results, the oxidase-like activity of SA-PtNPs originates mainly from the formation of O2•- during the reaction.

Several experiments have been carried out to investigate the contribution of SA in SA-PtNPs. First of all, we found that bare PtNPs show distinct aggregation in solution, while the SA-PtNPs exhibited excellent colloidal stability when SA was introduced into the preparation process (Supplementary Figure 8). Compared with bare platinum without any protective agent, SA-PtNPs obtain a much higher oxidase-like activity that implies the improvement effect with the introduction of SA (Figure 3A). As is generally known, the stability of PtNPs against aggregation under different harsh conditions is crucial to the extension of the practical application. To this end, SA-PtNPs were exposed to several pH values (2–12) for 2 h to explore the effect of pH on the oxidase activity. As shown in Figure 3B, the oxidase-like activity of SA-PtNPs fluctuated slightly with the change of environment pH, implying excellent pH stability of SA-PtNPs. Furthermore, we found that SA-PtNPs possessed perfect oxidase-like activity even if they have been stored at room temperature for a long time (Figure 3C). The above results confirm that the contribution of SA in PtNPs preparation increases the oxidase-like activity and stability of PtNPs.

The phenolic hydroxyl groups in the OPC structure make it a good hydrogen donor and therefore, it has a strong antioxidant capacity (Shao et al., 2018). Owing to the scavenging ability of OPC to O2•- and the formation of O2•- in the SA-PtNPs-TMB system, it is speculated that OPC could inhibit the catalytic process. Thus, a colorimetric assay for OPC was established (Scheme 1).

Experimental results in Figure 4A indicated the A_450nm of the SA-PtNPs-TMB system gradually decreased as the concentration of OPC increases. When the concentration of OPC in the reaction system reached 1 mM, the oxidation of TMB by SA-PtNPs was drastically inhibited. These phenomena fully illustrate that OPC could distinctly inhibit the color response of the SA-PtNPs-TMB system, namely inhibiting the oxidase-like activity of SA-PtNPs. Linearity was obtained by plotting the value of ΔA_450nm and the logarithmic value of OPC concentration (Figure 4B). (ΔA_450nm=A₀-A_t, where A₀ and A_t are the absorbance of the reaction product at 450 nm in the inexistence and existence of OPC, respectively.) The range of linearity was from 4 to 32.5 μM, the relative standard deviation (RSD) is 0.6% for measuring 10 μM, and limit of detection (LOD) for OPC was 2.0 μM (ΔA_450nm=1.1791 lgC_OPC + 3.1941, r = 0.999). As shown in Supplementary Table 1, most reported methods use high-performance liquid chromatography or mass spectrometry technology to analyze PC or OPC and are time-consuming, expensive, and technically demanding. What's more, the proposed method showed a comparable sensitivity and LOD to those of the reported methods.

Based on this linear equation, the antioxidant capacity of OPC was also assessed. Before measurements, OPC extracted from grape seeds was centrifuged to remove any solid residue (Soxhlet extraction method, Supplementary Material). AA is a well-recognized substance with antioxidant capacity. In this study, the inhibitory effect of 1 mM AA on the SA-PtNPs-TMB system is defined as 1 U. Therefore, the antioxidant capacity of pure OPC was calculated to be 2.85 U/mg (Supplementary Material). More significantly, as shown in Table 2, the recoveries of the standard addition experiment in grape seeds ranged from 97.0 to 98.6% with RSD values of 0.5–3.4%. These results provide a new insight for antioxidant capacity and indicate the accuracy of this assay for OPC analysis.