Vancomycin-Stabilized Platinum Nanoparticles with Oxidase-like Activity for Sensitive Dopamine Detection

The development of efficient, reliable, and sensitive dopamine detection methods has attracted much attention. In this paper, vancomycin-stabilized platinum nanoparticles (Van-Ptn NPs, n = 0.5, 1, 2) were prepared by the biological template method, where n represented the molar ratio of vancomycin to Pt. The results show that Van-Pt2 NPs had oxidase-like activity and peroxidase-like activity, and the mechanism was due to the generation of reactive oxygen 1O2 and OH. Van-Pt2 NPs exhibited good temperature stability, storage stability, and salt solution stability. Furthermore, Van-Pt2 NPs had almost no cytotoxicity to A549 cells. More importantly, the colorimetric detection of DA in human serum samples was performed based on the oxidase-like activity of Van-Pt2 NPs. The linear range of DA detection was 10–700 μM, and the detection limit was 0.854 μM. This study establishes a rapid and reliable method for the detection of dopamine and extends the application of biosynthetic nanoparticles in the field of biosensing.


Introduction
Dopamine (DA) is a neurotransmitter that regulates a variety of physiological processes in the human body, such as emotion, memory, behavior, and learning [1,2].It can also affect the body's metabolism, such as appetite, sleep, temperature regulation, and exercise capacity.Abnormal dopamine secretion can lead to cognitive dysfunction, Parkinson's disease, anxiety, obesity, and so on [3].In addition, drug consumption can also lead to excessive secretion of DA, which leads to drug addiction.Thus, it is important to determine the concentration of DA.At present, the analytical methods for the detection of DA include colorimetric analysis [4], fluorescence analysis [5], electrochemical analysis [6], and photothermal analysis [7].Compared with other analysis methods, the colorimetric method has the advantages of simple operation, low cost, and good visibility.In colorimetric analysis, it is necessary to add natural enzymes to accelerate the reaction.Natural enzymes have attracted much attention due to their high catalytic activity, high selectivity, and high specificity.However, the application of natural enzymes is restricted by shortcomings such as difficult storage and easy inactivation [8].
Compared with natural enzymes, nanozymes have the advantages of easy storage, simple preparation, and easy control.At present, a large number of nanomaterials with oxidase-like and peroxidase-like activities have been developed, including metal oxide nanomaterials [9], carbon-based nanomaterials [10], metal-organic frameworks [11] and noble metal nanomaterials [12].Noble metal nanomaterials have a large specific surface area and unique D-layer electron orbitals, which can make reactants more inclined to adsorb on their surface and thus form reactive intermediates [13].Therefore, noble metal nanomaterials have the advantages of high catalytic activity and enzyme-like activity.Based on their enzyme-like activities, noble metal nanozymes can be used to construct economical DA detection platforms.Platinum nanoparticles (Pt NPs) are among the most effective catalysts due to their superior enzyme-like activity, good biocompatibility, and catalytic chemiluminescence properties, which can serve as mimic enzymes for oxidase, peroxidase, catalase, and superoxide dismutase [14].Wang et al. [15] synthesized an atomically dispersed diatomic active site nanozymes (FeN 3 /PtN 4 -single-atom nanozymes (SAzyme)) through a two-part pyrolysis process.This FeN 3 /PtN 4 -SAzyme showed good DA colorimetric detection performance.The linear detection range of DA was 1-10 µM, and the detection limit was 0.109 µM.Li et al. [16] prepared Pt/NH101-MIL-2 hybrid nanozymes with bimetallic catalytic centers by forming a coordination bond between Pt nanoparticles (Pt NPs) and -NH 2 on a metal-organic skeleton (MOF).The catalytic activity of Pt/NH 2 -MIL-101 was increased by 1.5 times, and the detection limit of DA molecule was only 0.42 µM by colorimetry.
However, nanozymes for DA detection are mainly synthesized via physical and chemical methods, resulting in nanozymes that are generally toxic, unstable, aggregated, and environmentally unfriendly.In the face of these problems, bioregulated synthesis using biological molecules has attracted much attention due to its capacity to control the structure of nanomaterials and improve biocompatibility.As an antibiotic, vancomycin has good water solubility.In addition, its own hydroxyl group and amino group can not only form hydrogen bonds with water but also form weak forces with Pt 2+ and Pt atoms so as to achieve the effects of a mineralizer and a stabilizer.However, the facile detection of DA by vancomycin-stabilized platinum nanoparticles has not been reported.
Herein, vancomycin-stabilized platinum nanoparticles (Van-Pt n NPs n = 0.5, 1, 2) were prepared by adding different amounts of K 2 PtCl 4 with vancomycin under the reducing agent.This was carried out to investigate the structure characterization, enzyme-like activity, catalytic kinetics, and catalytic mechanism of the synthesized Van-Pt n NPs.Among them, the Van-Pt 2 NPs showed good thermal stability with long storage time and storage stability.Based on the oxidase-like activity of Van-Pt 2 NPs, a simple and sensitive method for DA detection was developed.In addition, Van-Pt 2 NPs also exhibited good biocompatibility.The Van-Pt 2 NPs in this study provides a novel strategy for DA detection with great potential application.

Materials and Methods
The materials, synthetic method for Van-Pt n NPs (n = 0.5, 1, 2), material characterization, enzyme activity-testing experiments, cytocompatibility experiments, and DA assays are described in detail in the supporting information.

Preparation and Structure Analysis
The synthesis of Van-Pt n NPs can be preliminarily characterized by the disappearance or appearance of specific peaks.As shown in Figure 1A, K 2 PtCl 4 has two characteristic absorption peaks at 392 nm and 329 nm.After vancomycin was co-incubated with K 2 PtCl 4 for 12 h, NaBH 4 was added to reduce Pt 2+ to form Pt for 12 h, and the two characteristic absorption peaks of Pt 2+ disappeared.This is similar to the characterization of Pt NPs prepared by Li et al. [17].In addition, Figure 1B shows that with the increase in the molar ratio of vancomycin to K 2 PtCl 4 , the absorbance of the synthesized Van-Pt n NPs also increased and the color of the sample deepened.Among them, Van-Pt 2 NPs have the highest absorbance.The morphology of Van-Pt n NPs and the size of their particles were characterized by TEM [18].The particle sizes were 5.48 ± 0.6 nm for Van-Pt 0.5 NPs (Figure 1C,D), 5.15 ± 0.3 nm for Van-Pt 1 NPs (Figure 1E,F), and 5.63 ± 0.4 nm for Van-Pt 2 NPs (Figure 1G,H), respectively.Thus, there is no significant difference in particle size and morphology between Van-Pt n NPs at the three ratios.
Biomolecules 2023, 13, x FOR PEER REVIEW 3 of 13 characterized by TEM [18].The particle sizes were 5.48 ± 0.6 nm for Van-Pt0.5 NPs (Figure 1C,D), 5.15 ± 0.3 nm for Van-Pt1 NPs (Figure 1E,F), and 5.63 ± 0.4 nm for Van-Pt2 NPs (Figure 1G,H), respectively.Thus, there is no significant difference in particle size and morphology between Van-Ptn NPs at the three ratios.The catalysis of nanozymes is mainly carried out in aqueous solution.The states of nanomaterials are measured by DLS [19].As depicted in Figure 2A, the hydrodynamic sizes of Van-Pt 0.5 NPs, Van-Pt 1 NPs, and Van-Pt 2 NPs were 20.05 ± 0.43 nm, 21.07 ± 0.51 nm, and 18.78 ± 0.94 nm, respectively.As shown in Figure 2B, the zeta potentials of Van-Pt 0.5 NPs, Van The catalysis of nanozymes is mainly carried out in aqueous solution.The states of nanomaterials are measured by DLS [19].As depicted in Figure 2A, the hydrodynamic sizes of Van-Pt0.5 NPs, Van-Pt1 NPs, and Van-Pt2 NPs were 20.05 ± 0.43 nm, 21.07 ± 0.51 nm, and 18.78 ± 0.94 nm, respectively.As shown in Figure 2B, the zeta potentials of Van-Pt0.5 NPs, Van-Pt1 NPs, and Van-Pt2 NPs were −13.53 ± 2.66 mV, −21.22 ± 1.97 mV, and −21.35 ± 2.15 mV, respectively.Among them, Van-Pt2 NPs had the largest absolute zeta potential value, which was conducive to the better stability and dispersion of Van-Pt2 NPs in aqueous solution.Van-Pt2 NPs were used as a representative to characterize the elemental composition and valence states of Van-Pt2 NPs by XPS [20].Figure S1A has four elements: C, N, O, and Pt.The three elements-C, N, and O-were all derived from the biological templatevancomycin-and Pt was reduced from K2PtCl4.As shown in Figure S1B, the corresponding binding energies of Pt 4f7/2 and Pt 4f5/2 are 71.2 eV and 74.5 eV, respectively.This result was very similar to the characterization of highly dispersed Pt NPs on N-doped ordered mesoporous carbon prepared by Sheng et al. [21] Thus, the Pt 2+ in K2PtCl4 was reduced from +2 valence to 0 valence, which proved the successful preparation of Van-Pt2 NPs.
Figure S1C shows that C 1s binding energies are 284.6 eV, 286.3 eV, and 288.5 eV, which correspond to C-C [22], C-O [23], and C=O [24], respectively.The binding energy of N 1s in Figure S1D is 399.5 eV, which is consistent with the binding energy of N-C in nitrogen-doped graphene nanoribbon reported by Yamada Y et al. [25].The elements C and N are provided by vancomycin.Therefore, the successful recombination of vancomycin and Pt NPs was confirmed via XPS determination.According to the XRD patterns in Figure S1E, the diffraction angles of Van-Pt2 NPs are 39.87°, 46.26°, 67.68°, 81.43°, and 86.12°, which correspond to the (111), ( 200), ( 220), (311), and (222) crystal planes, respectively.Compared with the reference code of Pt 01-001-1194, it is found that Pt NPs in the synthesized Van-Pt2 NPs had a face-centered cubic crystal structure.In short, these results suggest the successful preparation of Van-Pt2 NPs.

Enzyme-Like Property
To measure whether our synthesized Van-Pt2 NPs have oxidase-like activity, we designed five sets of experiments with 3,3′,5,5′-tetramethylbenzidine (TMB) for the oxidation substrate: (1) TMB; (2) Van-Pt2 NPs; (3) TMB + Van-Pt2 NPs; (4) TMB + Van; (5) TMB + Pt NPs.According to Figure 3A, group (3) TMB + Van-Pt2 NPs has the largest absorbance as 0.98 at 652 nm.The color of the samples in the group (3) of TMB + Van-Pt2 NPs changed from transparent to blue due to the oxidation of TMB to oxTMB.However, the absorbance of other groups was much lower than that of group (3).Moreover, Van-Pt2 NPs had higher absorbance for TMB reaction than bare Pt NPs and vancomycin, indicating that Van-Pt 2 NPs were used as a representative to characterize the elemental composition and valence states of Van-Pt 2 NPs by XPS [20].Figure S1A has four elements: C, N, O, and Pt.The three elements-C, N, and O-were all derived from the biological template-vancomycin-and Pt was reduced from K 2 PtCl 4 .As shown in Figure S1B, the corresponding binding energies of Pt 4f 7/2 and Pt 4f 5/2 are 71.2 eV and 74.5 eV, respectively.This result was very similar to the characterization of highly dispersed Pt NPs on N-doped ordered mesoporous carbon prepared by Sheng et al. [21] Thus, the Pt 2+ in K 2 PtCl 4 was reduced from +2 valence to 0 valence, which proved the successful preparation of Van-Pt 2 NPs.
Figure S1C shows that C 1s binding energies are 284.6 eV, 286.3 eV, and 288.5 eV, which correspond to C-C [22], C-O [23], and C=O [24], respectively.The binding energy of N 1s in Figure S1D is 399.5 eV, which is consistent with the binding energy of N-C in nitrogen-doped graphene nanoribbon reported by Yamada Y et al. [25].The elements C and N are provided by vancomycin.Therefore, the successful recombination of vancomycin and Pt NPs was confirmed via XPS determination.According to the XRD patterns in Figure S1E, the diffraction angles of Van-Pt 2 NPs are 39.87 • , 46.26 • , 67.68 • , 81.43 • , and 86.12 • , which correspond to the (111), ( 200), ( 220), (311), and (222) crystal planes, respectively.Compared with the reference code of Pt 01-001-1194, it is found that Pt NPs in the synthesized Van-Pt 2 NPs had a face-centered cubic crystal structure.In short, these results suggest the successful preparation of Van-Pt 2 NPs.

Enzyme-Like Property
To measure whether our synthesized Van-Pt 2 NPs have oxidase-like activity, we designed five sets of experiments with 3,3 ,5,5 -tetramethylbenzidine (TMB) for the oxidation substrate: (1) TMB; (2) Van-Pt 2 NPs; (3) TMB + Van-Pt 2 NPs; (4) TMB + Van; (5) TMB + Pt NPs.According to Figure 3A, group (3) TMB + Van-Pt 2 NPs has the largest absorbance as 0.98 at 652 nm.The color of the samples in the group (3) of TMB + Van-Pt 2 NPs changed from transparent to blue due to the oxidation of TMB to oxTMB.However, the absorbance of other groups was much lower than that of group (3).Moreover, Van-Pt 2 NPs had higher absorbance for TMB reaction than bare Pt NPs and vancomycin, indicating that vancomycin modification enhanced the oxidase-like activity of Pt NPs.Thus, Van-Pt 2 NPs have oxidaselike activity.It should be noted that the reaction time was 5 min.He et al. [26] prepared chondroitin sulfate-modified platinum nanozyme (CS-Pt NPs) exhibiting enhanced oxidaselike activity.The oxidase-like activity of CS-Pt NPs can be ascribed to the formation of the O 2 center dot from the activation of dissolved O 2 .
vancomycin modification enhanced the oxidase-like activity of Pt NPs.Thus, Van-Pt2 NPs have oxidase-like activity.It should be noted that the reaction time was 5 min.He et al. [26] prepared chondroitin sulfate-modified platinum nanozyme (CS-Pt NPs) exhibiting enhanced oxidase-like activity.The oxidase-like activity of CS-Pt NPs can be ascribed to the formation of the O2 center dot from the activation of dissolved O2.  3B, the group (4) TMB + Van-Pt2 NPs + H2O2 has the largest absorbance as 1.18, which is much greater than that in group (3) TMB + Van-Pt2 NPs.In addition, the absorbance of group (1) TMB + H2O2 and group (2) Van-Pt2 NPs + H2O2 is close to 0. These results indicated that Van-Pt2 NPs had peroxidase-like activity.To test whether Van-Pt 2 NPs have the ability to oxidize different substrates, 1,2diaminobenzene (OPD) and 2,2 -azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS) were used as substrates instead of TMB.The following experiments were designed: (1) OPD; (2) Van-Pt 2 NPs; (3) Van-Pt 2 NPs + OPD.ABTS was also used as a substrate to measure.As shown in Figure 3D,E, OPD was oxidized to orange-red oxidized OPD (oxOPD) with characteristic peaks at 448 nm in group (3) Van-Pt 2 NPs + OPD, and ABTS was oxidized to blue oxidized ABTS (oxABTS) with characteristic peaks at 420 nm in group Van-Pt 2 NPs + ABTS.Thus, the synthesized Van-Pt 2 NPs have the ability to oxidize different substrates.

Optimal Catalytic Conditions and Catalytic Kinetics
Similar to native enzymes and other nanomaterial-based oxidase-like nanozymes, the catalytic activity of the prepared Van-Pt 2 NPs may be affected by external conditions such as pH and temperature.Therefore, we explored the optimal pH and temperature for oxidase-like activity of Van-Pt 2 NPs.According to Figure 4A, the oxidase-like activity at pH = 3 was set to 100%.When pH = 2 and pH = 4, the oxidase-like activity of Van-Pt 2 NPs decreased to 42.9% and 49.6%, respectively.Van-Pt 2 NPs in other pHs showed very low oxidase-like activity.Thus, Van-Pt 2 NPs had the highest oxidase-like activity at pH = 3.In Figure 4B, the activity of Van-Pt 2 NPs reached its highest value at 30 • C. The activity of Van-Pt 2 NPs decreased at other temperatures but remained at 69.6%.Thus, Van-Pt 2 NPs have the highest oxidase-like activity when the temperature is 30 • C. Therefore, the optimum conditions for the enzymatic activity of Van-Pt 2 NPs were pH = 3 and a temperature of 30 • C, and the subsequent experiments were performed under these conditions.
(2) Van-Pt2 NPs; (3) Van-Pt2 NPs + OPD.ABTS was also used as a substrate to measure.As shown in Figure 3D, E, OPD was oxidized to orange-red oxidized OPD (oxOPD) with characteristic peaks at 448 nm in group (3) Van-Pt2 NPs + OPD, and ABTS was oxidized to blue oxidized ABTS (oxABTS) with characteristic peaks at 420 nm in group Van-Pt2 NPs + ABTS.Thus, the synthesized Van-Pt2 NPs have the ability to oxidize different substrates.

Optimal Catalytic Conditions and Catalytic Kinetics
Similar to native enzymes and other nanomaterial-based oxidase-like nanozymes, the catalytic activity of the prepared Van-Pt2 NPs may be affected by external conditions such as pH and temperature.Therefore, we explored the optimal pH and temperature for oxidase-like activity of Van-Pt2 NPs.According to Figure 4A, the oxidase-like activity at pH = 3 was set to 100%.When pH = 2 and pH = 4, the oxidase-like activity of Van-Pt2 NPs decreased to 42.9% and 49.6%, respectively.Van-Pt2 NPs in other pHs showed very low oxidase-like activity.Thus, Van-Pt2 NPs had the highest oxidase-like activity at pH = 3.In Figure 4B, the activity of Van-Pt2 NPs reached its highest value at 30 °C.The activity of Van-Pt2 NPs decreased at other temperatures but remained at 69.6%.Thus, Van-Pt2 NPs have the highest oxidase-like activity when the temperature is 30 °C.Therefore, the optimum conditions for the enzymatic activity of Van-Pt2 NPs were pH = 3 and a temperature of 30 °C, and the subsequent experiments were performed under these conditions.
Therefore, the catalytic reaction kinetics of Van-Pt2 NPs were characterized to explore the oxidase-like activity.The reaction kinetics of the nanozyme was determined by varying the TMB concentration.The test data were then analyzed using the Lineweaver-Burk double reciprocal to obtain the data plot of Figure 4C, D. The Michaelis constant Km (mM) and the maximum reaction rate Vmax (Ms −1 ) were then calculated from the double reciprocal line.The standard equation for TMB was y = 0.01899x + 0.01512 (R 2 = 0.9971), the substrate was TMB, the substrate concentration was 0.04-0.4mM, and the Km and Vmax values of Van-Pt2 NPs were 1.256 mM and 66.138 × 10 −8 Ms −1 , respectively.It can be seen from Table 1 that Van-Pt2 NPs have obvious advantages over other nanozymes, such as Mn0.6Co0.4OMS [27], with a Vmax value of 7.62 × 10 −8 Ms −1 .This also indicates that Van-Pt2 NPs have excellent oxidase-like activity, which is beneficial for their catalytic reactions.Therefore, the catalytic reaction kinetics of Van-Pt 2 NPs were characterized to explore the oxidase-like activity.The reaction kinetics of the nanozyme was determined by varying the TMB concentration.The test data were then analyzed using the Lineweaver-Burk double reciprocal to obtain the data plot of Figure 4C,D.The Michaelis constant K m (mM) and the maximum reaction rate V max (Ms −1 ) were then calculated from the double reciprocal line.The standard equation for TMB was y = 0.01899x + 0.01512 (R 2 = 0.9971), the substrate was TMB, the substrate concentration was 0.04-0.4mM, and the K m and V max values of Van-Pt 2 NPs were 1.256 mM and 66.138 × 10 −8 Ms −1 , respectively.It can be seen from Table 1 that Van-Pt 2 NPs have obvious advantages over other nanozymes, such as Mn 0.6 Co 0.4 O MS [27], with a V max value of 7.62 × 10 −8 Ms −1 .This also indicates that Van-Pt 2 NPs have excellent oxidase-like activity, which is beneficial for their catalytic reactions.

Stability of Van-Pt 2 NPs
After successful preparation of Van-Pt 2 NPs nanozymes and verification of their biomimetic properties, the stability of Van-Pt 2 NPs was tested, including temperature stability, storage stability, and salt solution stability.Figure 5A is the temperature stability of Van-Pt 2 NPs.After incubation for 120 min in the range of 10 • C to 80 • C, Van-Pt 2 NPs showed good catalytic performance in the range of 30 • C to 60 • C, while the catalytic performance increased with temperature in the range of 10 • C to 30 • C.After 60 • C, the catalytic performance gradually decreased to 78.6%.The oxidase-like activity of Van-Pt 2 NPs maintained at a high level.Therefore, it can be concluded that Van-Pt 2 NPs have good temperature stability.
Figure 5B shows the storage time stability test of Van-Pt 2 NPs.With the increase in storage time, the oxidase-like activity of Van-Pt 2 NPs remained around 100% with a fluctuation range of 97% to 105%.Therefore, the Van-Pt 2 NPs have good storage stability.Figure 5C shows that the hydrodynamic size of Van-Pt 2 NPs slowly grows by 3 nm with increasing storage time.Therefore, the synthesized Van-Pt 2 NPs showed good storage stability.The catalytic properties of Van-Pt 2 NPs were tested in different buffer solutions.The absorbance of Van-Pt 2 NPs in HAc-NaAc buffer solution, H 3 BO 3 buffer solution, and PBS buffer solution was 1.67, 1.25, and 0.79 in Figure 5D, respectively.The absorbance of Van-Pt 2 NPs was highest in HAc-NaAc buffer solution.Therefore, Van-Pt 2 NPs have the best oxidase-like activity in HAc-NaAc buffer solution.

Catalytic Mechanism of Van-Pt 2 NPs
The oxidation of TMB is closely related to the species of reactive oxygen species (ROS).Therefore, it is necessary to explore the kinds of ROS produced in the oxidase-like reaction of Van-Pt 2 NPs.Different ROS inhibitors-sodium azide (NaN 3 ), isopropyl alcohol (IPA), ethylenediaminetetraacetic acid disodium salt (EDTA-2Na), and 1,4-Benzoquinone (BQ)-quench or inhibit 1 O 2 , OH, h + , and O 2 − , respectively.In order to explore the types of ROS generated in the oxidase-like activity reaction of Van-Pt 2 NPs, we set up five groups experiments: (1) Van-Pt 2 NPs + TMB; (2) Van-Pt 2 NPs + TMB + NaN 3 ; (3) Van-Pt 2 NPs + TMB + IPA; (4) Van-Pt 2 NPs + TMB + EDTA-2Na; (5) Van-Pt 2 NPs + TMB + BQ. Figure 6 shows that the absorbance of the reaction decreased by 90.7% after adding NaN 3 to the Van-Pt 2 NPs + TMB system.After adding IPA, the absorbance decreased by 21.3%.After the addition of BQ and EDTA-2Na, the absorbance decreased by 1.8% and 6.2%, respectively.These results indicated that ROS produced by the oxidase-like activity of Van-Pt 2 NPs are mainly 1 O 2 , containing a small amount of •OH without h + and O 2 − .Thus, the catalytic mechanism should be as follows: dissolved O 2 was adsorbed and decomposed on the surface of Van-Pt 2 NPs, and then the subsequently formed 1 O 2 and a small amount of OH by O 2 can react with the substrate, thereby exerting the oxidase-like activity of Van-Pt 2 NPs.

Catalytic Mechanism of Van-Pt2 NPs
The oxidation of TMB is closely related to the species of reactive oxygen species (ROS).Therefore, it is necessary to explore the kinds of ROS produced in the oxidase-like reaction of Van-Pt2 NPs.Different ROS inhibitors-sodium azide (NaN3), isopropyl alcohol (IPA), ethylenediaminetetraacetic acid disodium salt (EDTA-2Na), and 1,4-Benzoquinone (BQ)-quench or inhibit 1 O2, OH, h + , and O2 − , respectively.In order to explore the types of ROS generated in the oxidase-like activity reaction of Van-Pt2 NPs, we set up five groups experiments: (1) Van-Pt2 NPs + TMB; (2) Van-Pt2 NPs + TMB + NaN3; (3) Van-Pt2 NPs + TMB + IPA; (4) Van-Pt2 NPs + TMB + EDTA-2Na; (5) Van-Pt2 NPs + TMB + BQ. Figure 6 shows that the absorbance of the reaction decreased by 90.7% after adding NaN3 to the Van-Pt2 NPs + TMB system.After adding IPA, the absorbance decreased by 21.3%.After the addition of BQ and EDTA-2Na, the absorbance decreased by 1.8% and 6.2%, respectively.These results indicated that ROS produced by the oxidase-like activity of Van-Pt2 NPs are mainly 1 O2, containing a small amount of •OH without h + and O2 − .Thus, the catalytic mechanism should be as follows: dissolved O2 was adsorbed and decomposed on the surface of Van-Pt2 NPs, and then the subsequently formed 1 O2 and a small amount of OH by O2 can react with the substrate, thereby exerting the oxidase-like activity of Van-Pt2 NPs.

Detection of DA
Van-Pt 2 NPs have the ability to oxidize to TMB, and DA can reduce blue oxTMB to colorless TMB.Thus, we were able to construct a method to detect DA using the excellent oxidase-like activity of Van-Pt 2 NPs.The experimental system is Van-Pt 2 NPs + TMB + DA.As the concentration of dopamine (0-5 mM) gradually increased in the reaction system, the absorption intensity of the mixed solution at 652 nm gradually decreased (Figure 7A).At the same time, as shown in Figure 7B, ∆A (∆A = A blank − A DA ) was linear with C DA when the concentration of DA varied from 10 to 700 µM.The DA standard curve equation was established in the detection range of 10-700 µM: Y = 0.0713 + 0.53498 × C DA (R 2 = 0.9971), the slope and intercept were 0.53498 and 0.0713, respectively, and the limit of detection was 0.854 µM (LOD = 3σ/K).As shown in Table 2, compared with the reported SiW 9 Co 3 [32] and h-CuS NCs [33] nanozymes, the detection range was 5.38-108 µM and 2-150 µM, respectively, and the detection limit was 5.38 µM and 1.67 µM, respectively.Therefore, Van-Pt 2 NPs have a wider detection range and better sensitivity.

Detection of DA
Van-Pt2 NPs have the ability to oxidize to TMB, and DA can reduce blue oxTMB to colorless TMB.Thus, we were able to construct a method to detect DA using the excellent oxidase-like activity of Van-Pt2 NPs.The experimental system is Van-Pt2 NPs + TMB + DA.As the concentration of dopamine (0-5 mM) gradually increased in the reaction system, the absorption intensity of the mixed solution at 652 nm gradually decreased (Figure 7A).At the same time, as shown in Figure 7B, ΔA (ΔA = Ablank − ADA) was linear with CDA when the concentration of DA varied from 10 to 700 μM.The DA standard curve equation was established in the detection range of 10-700 μM: Y = 0.0713 + 0.53498 × CDA (R 2 = 0.9971), the slope and intercept were 0.53498 and 0.0713, respectively, and the limit of detection was 0.854 μM (LOD = 3σ/K).As shown in Table 2, compared with the reported SiW9Co3 [32] and h-CuS NCs [33] nanozymes, the detection range was 5.38-108 μM and 2-150 μM, respectively, and the detection limit was 5.38 μM and 1.67 μM, respectively.Therefore, Van-Pt2 NPs have a wider detection range and better sensitivity.Other substances may affect the accuracy of DA detection, so it is necessary to test the anti-interference performance of Van-Pt 2 NPs.The interfering agents included Mg 2+ ,alanine (Ala), phenylalanine (Phe), leucine (Leu), glycine (Gly), proline (Pro), glutamic acid (Glu), maltose (Mal), lactose (Lac), and fructose (Fru).The concentration of DA was 3.33 mM, and the concentration of interfering agent was 10 mM. Figure 7C shows that the absorbance ∆A of the solution reached 0.80 when DA was added.When other interfering agents were added, the maximum absorbance ∆A of the solution was only 0.14.Therefore, Van-Pt 2 NPs had anti-interference performance for DA detection.
Based on the accuracy and selectivity of Van-Pt 2 NPs, the recovery rate of DA in real samples was studied.The recovery rate of the sample was calculated using the DA standard curve equation, and the recovery rate of DA was 99-110% (Table 3).Other substances may affect the accuracy of DA detection, so it is necessary to test the anti-interference performance of Van-Pt2 NPs.The interfering agents included Mg 2+ ,alanine (Ala), phenylalanine (Phe), leucine (Leu), glycine (Gly), proline (Pro), glutamic acid (Glu), maltose (Mal), lactose (Lac), and fructose (Fru).The concentration of DA was 3.33 mM, and the concentration of interfering agent was 10 mM. Figure 7C shows that the absorbance ΔA of the solution reached 0.80 when DA was added.When other interfering agents were added, the maximum absorbance ΔA of the solution was only 0.14.Therefore, Van-Pt2 NPs had anti-interference performance for DA detection.
Based on the accuracy and selectivity of Van-Pt2 NPs, the recovery rate of DA in real samples was studied.The recovery rate of the sample was calculated using the DA standard curve equation, and the recovery rate of DA was 99-110% (Table 3).
Table 3. Detects the recovery of DA in tap water and sea water.

Conclusions
In summary, we successfully synthesized a kind of nanozyme with high activity of oxidase-like and peroxidase-like activity via biological template method.The synthesized Van-Pt 2 NPs had good thermal stability and storage time stability.The catalytic kinetics of Van-Pt 2 NPs conformed to the typical Michaelis-Menten equation.Moreover, a simple, fast, and reliable method for DA detection was established based on the oxidase-like activity of Van-Pt 2 NPs.The detection range was 10-700 µM, and the detection limit was 0.854 µM.In addition, the MTT assay showed that Van-Pt 2 NPs and vancomycin almost had no cytotoxicity to A549 cells.Therefore, we synthesized Van-Pt 2 NPs with multiple functionalities, good stability, and good biocompatibility.

Supplementary Materials:
The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/biom13091312/s1:Electronic Supporting Information (ESI) is available: Preparation of Van-Pt n NPs and Pt NPs; Enzyme-like activity assay; Optimal conditions; Catalytic kinetics; Stability test; Activity mechanism; Dopamine detection; Figure S1: XPS and XRD characterization; Figure S2: Biocompatibility test.References [36,37] are cited in the supplementary materials.

Figure 3 .
Figure 3. (A) Oxidase-like activity characterization, t = 5 min; (B) characterization of peroxidase-like activity, t = 2 min; (C) comparison of oxidase-like activity of Van-Pt n NPs (the reaction time was 5 min); characterization of substrate (D) OPD and (E) ABTS.To test whether Van-Pt 2 NPs have peroxidase-like activity, we designed four groups of experiments: (1) TMB + H 2 O 2 ; (2) Van-Pt 2 NPs + H 2 O 2 ; (3) TMB + Van-Pt 2 NPs; (4) TMB + Van-Pt 2 NPs + H 2 O 2 .It should be noted that the reaction time was 2 min.According to the experimental results in Figure 3B, the group (4) TMB + Van-Pt 2 NPs + H 2 O 2 has the largest absorbance as 1.18, which is much greater than that in group (3) TMB + Van-Pt 2 NPs.In addition, the absorbance of group (1) TMB + H 2 O 2 and group (2) Van-Pt 2 NPs + H 2 O 2 is close to 0. These results indicated that Van-Pt 2 NPs had peroxidase-like activity.The difference of absorbance of TMB + Van-Pt 2 NPs + H 2 O 2 was due to different reaction times; H 2 O 2 slowly breaks down into water and oxygen, which has a bad effect on its use.Thus, the oxidase-like activity of Van-Pt 2 NPs without using H 2 O 2 was carefully further studied.Van-Pt n NPs with different molar ratios (n = 0.5, 1, and 2) reacted with TMB.It can be seen from Figure 3C that group (4) Van-Pt 2 NPs + TMB has the highest absorbance, while the other groups demonstrate far lower absorbance value.Therefore, Van-Pt 2 NPs exhibited the highest peroxidase-like activity.

Figure 4 .
Figure 4. Optimal conditions for oxidase-like activity of Van-Pt2 NPs: (A) optimal pH; (B) optimal temperature; (C) catalytic kinetics of different TMB concentrations; (D) the double reciprocal plot of (C).

Figure 4 .
Figure 4. Optimal conditions for oxidase-like activity of Van-Pt 2 NPs: (A) optimal pH; (B) optimal temperature; (C) catalytic kinetics of different TMB concentrations; (D) the double reciprocal plot of (C).

Figure 5 .
Figure 5. Stability determination of Van-Pt 2 NPs: (A) temperature stability; (B) storage time stability; (C) hydrodynamic size variation with time; (D) the catalytic ability of Van-Pt 2 NPs in different buffer solution.

Figure 7 .
Figure 7. DA detection method by Van-Pt 2 NPs: (A) UV-vis absorption spectra; (B) linear fit plots of ∆A over different concentrations; (C) detection of DA selectivity.
-Pt 1 NPs, and Van-Pt 2 NPs were −13.53 ± 2.66 mV, −21.22 ± 1.97 mV, and −21.35 ± 2.15 mV, respectively.Among them, Van-Pt 2 NPs had the largest absolute zeta potential value, which was conducive to the better stability and dispersion of Van-Pt 2 NPs in aqueous solution.

Table 1 .
Comparison of the kinetic parameters Km and Vmax.

Table 1 .
Comparison of the kinetic parameters K m and V max .

Table 2 .
Comparison of DA detection range and detection limit of different materials.

Table 2 .
Comparison of DA detection range and detection limit of different materials.

Table 3 .
Detects the recovery of DA in tap water and sea water.