Platinum Palladium Bimetallic Nanozymes Stabilized with Vancomycin for the Sensitive Colorimetric Determination of L-cysteine

Many diseases in the human body are related to the level of L-cysteine. Therefore, it is crucial to establish an efficient, simple and sensitive platform for L-cysteine detection. In this work, we synthesized platinum palladium bimetallic nanoparticles (Van-Ptm/Pdn NPs) using vancomycin hydrochloride (Van) as a stabilizer, which exhibited high oxidase-like catalytic activity. In addition, the catalytic kinetics of the Van-Pt1/Pd1 NPs followed the typical Michaelis–Menten equation, exhibiting a strong affinity for 3,3′,5,5′-tetramethylbenzidine substrates. More importantly, we developed a simple and effective strategy for the sensitive colorimetric detection of L-cysteine using biocompatible Van-Pt1/Pd1 NPs. The detection limit was low, at 0.07 μM, which was lower than the values for many previously reported enzyme-like detection systems. The colorimetric method of the L-cysteine assay had good selectivity. The established method for the detection of L-cysteine showed promise for biomedical analysis.


Introduction
L-cysteine is one of the sulfur-containing α-amino acids with good water solubility.Meanwhile, L-cysteine is involved in the reduction process of cells and phospholipid metabolism in the liver.The intracellular concentration of L-cysteine is usually around 30-200 µM [1].High or low levels of L-cysteine in the body can cause diseases [2].Therefore, it is important to establish an efficient, reliable and sensitive L-cysteine assay platform with which to detect its concentration.Currently, many methods have been reported for the detection of L-cysteine, including electrochemical methods, high-performance liquid chromatography, spin photometric methods, fluorescence detection and colorimetric detection [3][4][5][6].However, many detection methods are limited in terms of their wide application by their cost, detection time, toxicity and environmental hazards.
In recent years, colorimetric detection has been considered as a promising method for the detection of L-cysteine due to its simplicity of operation and good visualization [7].Many natural enzymes have been widely used in colorimetric assays.For example, Huang et al. [8] used the peroxidase activity of fig protease for the colorimetric assay of L-cysteine.The application of natural enzymes was limited due to shortcomings such as their complex extraction process, fallibility and cost.
More recently, artificial enzymes with enzyme-like activities have been developed.Since the first study of Fe 3 O 4 nanomaterials for peroxidase-like activity in 2007 [9], nanozyme-based colorimetric sensing platforms have contributed to the rapid development of the diagnostic and bioanalytical fields.A variety of nanozymes including noble metals [10], metal oxides [11,12], carbon-based materials [13,14] and other nanomaterials [15][16][17][18] have been reported.Pandey et al. [19] reported the structural characterization of noble metal monometallic, bimetallic and trimetallic nanoparticles, in addition to evaluating their biocatalytic activity for the non-enzymatic sensing of glucose.In particular, artificial nanozymes composed of noble metal nanoparticles have a wide range of applications due to their high interfacial stability, easy preparation and modification [20], as in the case of Pt nanotubes [21], gold nanoparticles [22] and Pd nanoparticles.Compared with monometallic nanoparticles, bimetallic nanoparticles with synergistic effects have received widespread attention because of their higher catalytic activity [23,24].Jang et al. [25] reported a TiO 2 -loaded Pt-Pd bimetallic model catalyst.Compared with the monometallic catalyst, the d electrons of Pt-Pd bimetallic nanoparticles were transferred from Pt 5d to Pd 4d upon alloying and the orbital hybridization and electronic state broadening of Pt and Pd.This led to a significant improvement in the catalytic performance of the bimetallic Pt-Pd catalyst.The metals Pt and Pd are both face-centered, cubic-structured metals with similar lattice constants; thus, they are more likely to form a homogeneous alloy.When Pt and Pd form an alloy, the coupling between the metals can improve their catalytic performance.Jin et al. [26] reported a Pd-Pt bimetallic alloy nanowire that exhibited excellent oxidase activity in an acidic environment.
Despite their small size, noble metal nanoparticles have a large specific surface area and high catalytic activity.But they tend to aggregate easily in solutions, leading to reduced activity.Therefore, the introduction of various carriers to stabilize small nanoparticles is considered one of the most effective ways to improve the catalytic activity and stability of enzyme mimics [27].Among the nanoparticles, the carriers usually serve as a backbone to widely disperse and stabilize the active components, such as inorganic mesoporous silica [28], polymeric carriers [29] and polysaccharides, etc. [30].Because of their good biocompatibility and easy modification, peptides have become a good means of modification on the surface of nanomaterials [31].Vancomycin hydrochloride (Van), as an antibacterial peptide, is a glycopeptide antibiotic with a molecular weight of 1486 and has great potential for stabilizing precious metal nanoparticles.
In this study, we synthesized platinum palladium bimetallic nanoparticles (Van-Pt m /Pd n NPs, m = 1, 2; n = 1, 2) using Van as a biological template for the first time.The particle size of the Van-Pt m /Pd n NPs was around 5 nm.A high catalytic activity of the Van-Pt 1 /Pd 1 NPs was achieved by exploring the preparation method and the molar ratio of platinum and palladium.Based on the Van-Pt 1 /Pd 1 NPs' oxidase-like and peroxidaselike enzymatic activity, we developed a simple and effective colorimetric method for the determination of L-cysteine with a low detection limit, a wide detection range and good selectivity.Importantly, the use of Van may reduce the toxicity of noble metals, which may offer the possibility for the wide application of noble metal nanozymes.

Synthesis of Van-Pt m /Pd n Nanoparticles
(a) A total of 73 µL of Van solution (10 mM) was added to a 2 mL polyethylene (PE) tube, and then 98 µL of K 2 PtCl 4 solution (10 mM) was added.The solution was incubated at 25 The absorbance in the wavelength range of 200-800 nm was measured with a UV-vis spectrophotometer.The morphology was photographed using transmission electron microscopy (TEM); the crystal structure of the nanoparticles was characterized using an X-ray diffraction (XRD) meter with a diffraction ratio of 10 • -90 • ; the elements and valence states of the nanoparticles were determined via X-ray photoelectron spectroscopy (XPS); and the hydrodynamic size and zeta potential were determined with a Zetasizer Nano-ZS90.

Activity of Van-Pt 1 /Pd 1 Nanoparticles
The oxidase-like activity of the Van-Pt 1 /Pd 1 nanoparticles was determined by measuring the oxidized TMB.A total of 200 µL of Van-Pt 1 /Pd 1 nanoparticles (C Pt = 0.45 mM) was added to a 2 mL PE tube.Then, 300 µL of 0.2 M HAc-NaAc solution (pH = 3) was added, and 1000 µL of 0.2 M HAc-NaAc solution containing 0.6 mM TMB was added.The samples were incubated in a constant-temperature mixer at 25 • C and 600 rpm for 5 min.The absorbance was measured using a UV-vis spectrophotometer.In addition, the relative activity of the Van-Pt 1 /Pd 1 NPs was determined at different pHs (pH = 1-12) and temperatures (5-65 • C).The samples added to the PE tubes were varied according to the experimental requirements.
The peroxidase-like activity of the Van-Pt 1 /Pd 1 nanoparticles was determined by assaying the oxidized TMB produced under hydrogen peroxide conditions.200 µL of Van-Pt 1 /Pd 1 nanoparticles (C Pt = 0.45 mM) were added to a 2 mL PE tube.Then, 300 µL, 0.2 M of HAc-NaAc solution (pH = 3) was added; 1000 µL of 0.2 M HAc-NaAc solution containing 0.6 mM TMB was added, 100 µL, 0.03 M H 2 O 2 solution was added.The samples were incubated in a constant temperature mixer at 25 • C and 600 rpm for 2 min.The absorbance was measured by UV-vis spectrophotometer.

Catalytic Kinetics of Van-Pt 1 /Pd 1 Nanoparticles
In total, 200 µL of Van-Pt 1 /Pd 1 nanoparticles (C Pt = 0.45 mM) was added to a 2 mL PE tube.Then, we added 0.2 M of HAc-NaAc solution (pH = 3) and 0.2 M HAc-NaAc solution containing 0.6 mM TMB.The absorbance was measured using a UV-vis spectrophotometer.The amount of buffer solution was 1200-300 µL at 100 µL intervals, and the amount of buffer solution containing TMB was 100-1000 µL at 100 µL intervals.The total amount of liquid in the PE tube was 1500 µL.The affinity for the substrate and the maximum rate of the catalytic reaction during enzyme catalysis was studied using Formula (1) [32].
Here, V m is the maximum reaction rate; [S] is the substrate concentration; and K m is the Michaelis-Menten constant.

The Mechanism of Oxidase-Like Activity
The types of reactive oxygen species (ROS) produced during catalysis were studied by adding different ROS inhibitors to the solution.A total of 200 µL of Van-Pt 1 /Pd 1 NPs (C Pt = 0.45 mM) was added to a 2 mL PE tube.Then, 1000 µL of 0.2 M HAc-NaAc solution containing 0.6 mM TMB was added, and 200 µL of different solutions of reactive oxygen inhibitor solutions (10 mM) was added.The samples were incubated in a constant-temperature mixer at 30 • C and 600 rpm for 5 min.The absorbance was measured using a UV-vis spectrophotometer.The ROS inhibitors were p-benzoquinone (BQ), sodium nitride (NaN 3 ), isopropyl alcohol (IPA) and disodium ethylenediaminetetraacetate (EDTA-2Na), respectively.

Detection of L-cysteine Using Van-Pt 1 /Pd 1 Nanoparticles
A total of 50 µL of Van-Pt 1 /Pd 1 nanozymes (C Pt = 0.45 mM) was added to a 2 mL PE tube, followed by 1000 µL of 0.2 M, pH = 3 HAc-NaAc solution containing 0.6 mM TMB, and then 200 µL of aqueous L-cysteine solution at different concentrations.Then, the tube was placed in a constant-temperature reaction at 30 • C and 600 rpm for 5 min.Finally, the UV-vis spectrum was measured.The standard curve of the assay was obtained using the difference in absorbance versus concentration.The real samples were replaced with different samples with different concentrations of L-cysteine containing the spiked amount, and the recoveries were calculated according to the spiked recovery Formula (2) [33].
A i * is the theoretical absorbance, and A i is the actual absorbance.

Biocompatibility Test
The biocompatibility of the nanozymes was determined using the MTT method.First, cells were added to 96-well plates and incubated in a cell incubator for 24 h.Then, samples (C samples = 12.5-200 µg/mL) including Van and Van-Pt 1 /Pd 1 NPs were added to the 96-well tissue culture plates and incubated for 24 h.After that, MTT (100 µL, 500 µg/mL) was added.After 4 h, the MTT solution was removed, and DMSO solution was added.Finally, the absorbance of the 96-well plates was measured using a microplate reader.

Characterization of Van-Pt m /Pd n Nanoparticles
As shown in Figure 1A, a UV-vis spectrometer was used to scan the Van-Pt m /Pd n nanoparticles within 250-700 nm.The characteristic bands of Pt 2+ were at 392 nm and 329 nm, and Pd 2+ had a distinct characteristic absorption band at 420 nm.The Van-Pt m /Pd n NPs had no obvious characteristic absorption bands of Pt 2+ or Pd 2+ , which may be because Pt 2+ and Pd 2+ were reduced to Pt and Pd, respectively.The mere absence of Pt 2+ and Pd 2+ transitions in the UV-vis spectra suggested that Van-Pt m /Pd n nanoparticles may have formed.In addition, the color of the Van-Pt m /Pd n NPs was brown (Figure 1B), similar to the color of previously reported Pt/Pd NPs [34].All these results indicated the successful synthesis of Van-Pt m /Pd n NPs.As shown in Figure 2A-H, the particle sizes of the Van-Pd1-Pt2 NPs, Van-Pt2-Pd1 NPs, Van-Pt2/Pd1 NPs and Van-Pt1/Pd1 NPs were 5.3 ± 0.2 nm, 4.8 ± 0.6 nm, 5.7 ± 0.4 nm and 5.5 ± 0.5 nm, respectively.There was no significant difference in the particle size of the nanoparticles using the three synthesis methods.The Van-Pt2-Pd1 NPs and Van-Pd1-Pt2 NPs had a slight degree of aggregation.The Van-Pt2/Pd1 NPs and Van-Pt1/Pd1 NPs had better dispersion.The size of the Van-Ptm/Pdn NPs was very small.The aggregation of the Van-Pt2-Pd1 NPs and Van-Pd1-Pt2 NPs may be due to the long reaction time.As shown in Figure 2A-H, the particle sizes of the Van-Pd 1 -Pt 2 NPs, Van-Pt 2 -Pd 1 NPs, Van-Pt 2 /Pd 1 NPs and Van-Pt 1 /Pd 1 NPs were 5.3 ± 0.2 nm, 4.8 ± 0.6 nm, 5.7 ± 0.4 nm and 5.5 ± 0.5 nm, respectively.There was no significant difference in the particle size of the nanoparticles using the three synthesis methods.The Van The catalytic reaction of the nanoparticles was carried out in aqueous solutions, the hydrodynamic size and zeta potential of nanoparticles affect their catalytic activity.As shown in Figure 3A, the hydrodynamic sizes of the Van-Pd 1 -Pt 2 NPs, Van-Pt 2 -Pd 1 NPs, Van-Pt 2 /Pd 1 NPs and Van-Pt 1 /Pd 1 NPs were 36.9 ± 4.1 nm, 34.2 ± 1.6 nm, 18.2 ± 1.0 nm and 16.1 ± 1.2 nm, respectively.They were slightly larger than those observed using TEM.The reason for this is that the water molecules form a thin water film around the nanoparticles in solution, resulting in a larger hydrodynamic size than that observed with TEM [35].Compared with the Van-Pd 1 -Pt 2 NPs and Van-Pt 2 -Pd 1 NPs, the Van-Pt 2 /Pd 1 NPs and Van-Pt 1 /Pd 1 NPs prepared using the one-pot method exhibited a smaller hydrodynamic size, which may be due to the dispersion performance of the Van-Pt 2 /Pd 1 NPs and Van-Pt 1 /Pd 1 NPs.In addition, the zeta potentials of the Van-Pt m /Pd n NPs were also determined (Figure 3B).The zeta potentials of the Van-Pd 1 -Pt 2 NPs, Van-Pt 2 -Pd 1 NPs, Van-Pt 2 /Pd 1 NPs and Van-Pt 1 /Pd 1 NPs in aqueous solution were −28.5 ± 2.1 mV, −29.5 ± 3.2 mV, −18.9 ± 2.5 mV and −20.6 ± 2.5 mV, respectively.The absolute values of the zeta potentials of all four nanoparticles were greater than 18 mV.Their zeta potential favored their good stability and maintenance of catalytic activity in the solution state.reason for this is that the water molecules form a thin water film around the nanoparticles in solution, resulting in a larger hydrodynamic size than that observed with TEM [35].
Compared with the Van-Pd1-Pt2 NPs and Van-Pt2-Pd1 NPs, the Van-Pt2/Pd1 NPs and Van-Pt1/Pd1 NPs prepared using the one-pot method exhibited a smaller hydrodynamic size, which may be due to the dispersion performance of the Van-Pt2/Pd1 NPs and Van-Pt1/Pd1 NPs.In addition, the zeta potentials of the Van-Ptm/Pdn NPs were also determined (Figure 3B).The zeta potentials of the Van-Pd1-Pt2 NPs, Van-Pt2-Pd1 NPs, Van-Pt2/Pd1 NPs and Van-Pt1/Pd1 NPs in aqueous solution were −28.5 ± 2.1 mV, −29.5 ± 3.2 mV, −18.9 ± 2.5 mV and −20.6 ± 2.5 mV, respectively.The absolute values of the zeta potentials of all four nanoparticles were greater than 18 mV.Their zeta potential favored their good stability and maintenance of catalytic activity in the solution state.The catalytic activity of nanozymes is also closely related to their preparation methods and reaction conditions.In order to obtain Van-Pt m /Pd n NPs with an excellent catalytic performance, we prepared nanoparticles with different metal ratios and different synthesis methods.Figure 3C shows that all the Van-Pt m /Pd n NPs had catalytic activity.Among them, the Van-Pt 1 /Pd 1 NPs prepared using the one-pot method exhibited the highest catalytic ability for TMB oxidation.This indicated that the nanoparticles prepared using the one-pot method had good catalytic activity.In this case, the reaction time of the Van-Pt 1 /Pd 1 NPs prepared using the one-pot method was 12 h, while the reaction time of the Van-Pt 1 -Pd 1 NPs and Van-Pd 1 -Pt 1 NPs prepared in a stepwise manner was 24 h, which caused the nanoparticles to become aggregated, leading to a decrease in the activity of the oxidase-like nanoparticles as compared to those obtained using the one-pot method.Meanwhile, we also compared other metal molar ratios of Van-Pt m /Pd n nanoparticles, i.e., m:n = 1:1, 1:2, 2:1, 1:5, 5:1, 1:10 and 10:1, as shown in Figure S1.The results showed that the Van-Pt 1 /Pd 1 NPs had the highest catalytic activity for TMB.
In short, among the three synthesis methods, the nanoparticles synthesized using the one-pot method had better dispersion and a smaller hydrodynamic size, which caused the nanoparticles to have more active sites; hence, the one-pot method was chosen to prepare the Pt Pd bimetallic nanoparticles.When comparing the catalytic activities of the nanozymes prepared with different molar ratios of Pt to Pd, the Van-Pt 1 /Pd 1 NPs showed the highest catalytic activity; thus, we chose the Van-Pt 1 /Pd 1 NPs for the subsequent experiments.
To further test our successful synthesis of Van-Pt m /Pd n NPs, we performed XPS and XRD characterizations of the Van-Pt 1 /Pd 1 NPs.The XPS spectra of the Van-Pt 1 /Pd 1 NPs showed five elements, C, N, O, Pt and Pd, as demonstrated in Figure 4A.Three elements, C, N and O, were derived from the biological template of Van, while Pt and Pd elements were reduced from K 2 PtCl 4 and Na 2 PdCl 4 , respectively.The binding energies at 71.2 eV and 74.7 eV corresponded to the Pt 4f 7/2 and Pt 4f 5/2 orbitals of the Pt elements in Figure 4B, respectively [36].This binding energy coincided with the binding energy of the 4f orbital of the Pt atom, which indicated that the Pt 2+ in K 2 PtCl 4 had been reduced to a Pt atom.In addition, the binding energies in Figure S2 are 284.6 eV, 286.2 eV and 288.6 eV, for which the corresponding chemical groups are C-C, C-O and C=O, respectively, and the C element was provided by Van [37][38][39].Figure 4C shows the XPS spectrum of Pd 3d.The binding energies of 335.0 eV and 340.5 eV correspond to Pd 3d 5/2 and Pd 3d 3/2 orbitals, respectively, which were consistent with the 3d orbital binding energy of Pd at a valency of 0 in the Pt/Pd alloy [40].Therefore, the successful loading of Pt/Pd alloy nanoparticles on the template of Van could be determined using XPS spectra, further proving our successful synthesis of Van-Pt 1 /Pd 1 NPs.The XRD results showed that the diffraction peaks appeared at 39.76 • , 46.24 • , 67.45 • , 81.28 • and 85.71 • , which correspond to the (1 1 1), (2 0 0), (2 2 0), (3 1 1) and (2 2 2) crystal planes of Pd and Pt, respectively (Figure 4D).Among the peaks, 39.76

Catalytic Activity of Van-Pt1/Pd1 NPs
To test the catalytic activity of the Van-Pt1/Pd1 NPs, we designed the following groups: 1TMB, 2Van-Pt1/Pd1 NPs, 3TMB + Van-Pt1/Pd1 NPs and 4TMB+ Pt1/Pd1 NPs.As shown in Figure 5A, the TMB + Van-Pt1/Pd1 NP group showed the highest characteristic absorption peak, and this characteristic absorption peak was provided by the oxidized TMB (oxTMB) [42,43].During this experiment, the absorbance of the TMB and Van-Pt1/Pd1 NPs at 652 nm was close to 0, while the absorbance of the TMB + Pt1/Pd1 NP group was only 26% that of the TMB + Van-Pt1/Pd1 NPs group.Therefore, the oxidase-like activity originated from the synthesized Van-Pt1/Pd1 NPs.

Catalytic Activity of Van-Pt 1/ Pd 1 NPs
To test the catalytic activity of the Van-Pt 1 /Pd 1 NPs, we designed the following groups: 1TMB, 2Van-Pt 1 /Pd 1 NPs, 3TMB + Van-Pt 1 /Pd 1 NPs and 4TMB+ Pt 1 /Pd 1 NPs.As shown in Figure 5A, the TMB + Van-Pt 1 /Pd 1 NP group showed the highest characteristic absorption peak, and this characteristic absorption peak was provided by the oxidized TMB (oxTMB) [42,43].During this experiment, the absorbance of the TMB and Van-Pt 1 /Pd 1 NPs at 652 nm was close to 0, while the absorbance of the TMB + Pt 1 /Pd 1 NP group was only 26% that of the TMB + Van-Pt 1 /Pd 1 NPs group.Therefore, the oxidase-like activity originated from the synthesized Van-Pt 1 /Pd 1 NPs.
Biomolecules 2023, 13, x 10 of 18 NPs + H2O2 group.Therefore, the Van-Pt1/Pd1 NPs had not only good oxidase-like activity but also peroxidase-like activity.The oxidase-like activity of the Van-Pt1/Pd1 NPs was very high; thus, the oxidase-like activity of the Van-Pt1/Pd1 NPs was investigated in the subsequent experiments.The oxidase-like activity of Van-Pt1/Pd1 NPs is influenced by external conditions, the main influencing factors being pH and temperature.In order to find the optimal conditions for enzyme catalysis, the oxidase-like activity of the Van-Pt1/Pd1 NPs at different pHs and temperatures was investigated.From Figure 5C, it can be seen that the Van-Pt1/Pd1 NPs had the best oxidase-like activity at pH = 3.The oxidase-like activity of the Van-Pt1/Pd1 NPs decreased at other pHs.Therefore, the optimal pH for the oxidase-like activity of the Van-Pt1/Pd1 NPs was 3. The pH affects the binding of the TMB with the Van-Pt1/Pd1 NPs.The substrate TMB should not be suitable for binding to the Van-Pt1/Pd1 NPs at a non-optimal pH, leading to a decrease in oxidase-like activity.Furthermore, Van-Pt1/Pd1 NPs should have the highest amount of reactive oxygen species at pH = 3.The effect of temperature on the enzyme activity was then explored under the conditions of the optimal pH.In Figure 5D, it can be seen that the highest value of the nanozymes' activity was reached at 30 °C.The activity of the nanozymes decreased at all other temperatures.However, the activity of the Van-Pt1/Pd1 NPs was maintained at a minimum of approximately The oxidase-like activity of Van-Pt 1 /Pd 1 NPs is influenced by external conditions, the main influencing factors being pH and temperature.In order to find the optimal conditions for enzyme catalysis, the oxidase-like activity of the Van-Pt 1 /Pd 1 NPs at different pHs and temperatures was investigated.From Figure 5C, it can be seen that the Van-Pt 1 /Pd 1 NPs had the best oxidase-like activity at pH = 3.The oxidase-like activity of the Van-Pt 1 /Pd 1 NPs decreased at other pHs.Therefore, the optimal pH for the oxidase-like activity of the Van-Pt 1 /Pd 1 NPs was 3. The pH affects the binding of the TMB with the Van-Pt 1 /Pd 1 NPs.The substrate TMB should not be suitable for binding to the Van-Pt 1 /Pd 1 NPs at a non-optimal pH, leading to a decrease in oxidase-like activity.Furthermore, Van-Pt 1 /Pd 1 NPs should have the highest amount of reactive oxygen species at pH = 3.The effect of temperature on the enzyme activity was then explored under the conditions of the optimal pH.In Figure 5D, it can be seen that the highest value of the nanozymes' activity was reached at 30 • C. The activity of the nanozymes decreased at all other temperatures.However, the activity of the Van-Pt 1 /Pd 1 NPs was maintained at a minimum of approximately 70%.Therefore, the optimal temperature for the Van-Pt 1 /Pd 1 NPs was 30 • C, and they had a wide temperature range of catalytic performance.Thus, we can define the optimal conditions for the enzymatic activity of Van-Pt 1 /Pd 1 NPs as pH = 3 and 30 • C. Subsequent experiments could be performed under these conditions.In addition, the catalytic activity of nanozymes is also related to nanoscale factors, such as their size, morphology and surface, which significantly affect their activity.Nayak et al. [44] assembled polyoxometalate (POM) (phosphotungstic acid (PTA)/phosphomolybdic acid (PMA)) nanoclusters and glucose oxidase (GOx) into microsphere structures, which facilitated the better diffusion of the reactants, intermediates and products due to the small size of the microspheres.This resulted in a 3-5-fold increase in the peroxidase-like activity of the PTA nanoclusters in the nanozyme microspheres.
To investigate the catalytic activity of the Van-Pt 1/ Pd 1 NPs, the kinetic characterization of the nanozymes was required.The reaction kinetics of the nanozymes were determined by varying the concentration of the substrate TMB [32].As shown in Figure 6A, the catalytic reaction followed the Michaelis-Menten equation in the concentration range of the substrate TMB (0.04-0.4 mM).As shown in Figure 6B, the standard equation was obtained as y = 0.00899x + 0.04109 (R 2 = 0.999) using the double-inverse data in Figure 6A.The K m and V max values of the Van-Pt 1 /Pd 1 NPs were 0.218 mM and 24.337 × 10 −8 Ms −1 , respectively.It can be seen from Table 1 that the K m of the Van-Pt 1 /Pd 1 NPs was smaller compared to the other nano-enzymes, such as ZIF-67 (13.69 mM), PdPt 3 -LNT NDs (0.263 mM), CeM (0.66 mM) and Cy-AuNCs (1.925 mM).Thus, the Van-Pt 1 /Pd 1 NPs had an excellent affinity for TMB.In addition, the Van-Pt 1 /Pd 1 NPs had a larger V max (24.337 × 10 −8 Ms −1 ) compared to the other nanomaterials, including the PdPt 3 -LNT NDs (2.88 × 10 −8 Ms −1 ), Pt-HMCN (15.4 × 10 −8 Ms −1 ), Pd 150 -PCRP NPs (15.58 × 10 −8 Ms −1 ) and N-CQDs (4.49 × 10 −8 Ms −1 ), which indicated that the Van-Pt 1 /Pd 1 NPs had a better catalytic effect compared to monometallic materials and non-precious metals.Therefore, the Van-Pt 1 /Pd 1 NPs had excellent oxidase-like activity, as shown not only by their larger V max but also by their excellent affinity with TMB.Meanwhile, the oxidase-like activity of the Van-Pt 1 /Pd 1 NPs was maintained at around 100% after one week of storage under ambient conditions, as shown in Figure 6C.Although the oxidase-like activity of the Van-Pt 1 /Pd 1 NPs fluctuated to some extent, the variation was within the range of 98-102%.In addition, after 120 min of incubation in the temperature range of 10-90 • C, the catalytic performance of the Van-Pt 1 /Pd 1 NPs remained around 90% in the range of 70-90 • C, as shown in Figure 6D.These findings indicated that the Van-Pt 1 /Pd 1 NPs had good stability in different pH conditions and temperature tolerance, and they had good catalytic activity in extreme environments.

Mechanism of the Oxidase-Like Activity of Van-Pt1/Pd1 NPs
The oxidation reaction of TMB based on Van-Pt1/Pd1 NPs is closely related to reactive oxygen species [20].Reactive oxygen species include as singlet oxygen ( 1 O2), hydroxyl radical (•OH), superoxide anion (O2• − ), etc.The mechanism of the oxidase-like activity of Van-Pt1/Pd1 NPs was investigated by adding different reactive oxygen species inhibitors, such as BQ, NaN3 and IPA, which have quenching effects on O2• − , 1 O2 and •OH, respectively.
To investigate the mechanism of the oxidase-like activity of Van-Pt1/Pd1 NPs, different experimental groups were set up, as shown in Figure 7A.It showed that the addition of NaN3 into the Van-Pt1/Pd1 NPs + TMB system (36%) had the greatest effect on the absorbance of the reaction, followed by the effect of IPA (77%), while BQ (104%) had the least effect and showed almost no difference compared with the control group of Van-Pt1/Pd1 NPs + TMB (100%).Therefore, the type of reactive oxygen species produced by the Van-Pt1/Pd1 NPs with oxidase-like activity was mainly 1 O2, containing a small amount of •OH with no O2• − production.To investigate the mechanism of the oxidase-like activity of Van-Pt 1 /Pd 1 NPs, different experimental groups were set up, as shown in Figure 7A.It showed that the addition of NaN 3 into the Van-Pt 1 /Pd 1 NPs + TMB system (36%) had the greatest effect on the absorbance of the reaction, followed by the effect of IPA (77%), while BQ (104%) had the least effect and showed almost no difference compared with the control group of Van-Pt 1 /Pd 1 NPs + TMB (100%).Therefore, the type of reactive oxygen species produced by the Van-Pt 1 /Pd 1 NPs with oxidase-like activity was mainly 1 O 2 , containing a small amount of •OH with no O 2 • − production.

L-cysteine Assay
Here, the cytotoxicity of the Van-Pt1/Pd1 NPs and Van was explored using the MTT assay.As shown in Figure 8A, when the concentration of the Van-Pt1/Pd1 NPs and Van was 200 µg/mL, the cell viability was maintained above 90%.This indicated that the Van-Pt1/Pd1 NPs were non-cytotoxic, as compared to previously reported Pt NPs and Pd NPs [52].Therefore, the Van-Pt1/Pd1 NPs synthesized using the bio-template method have good biocompatibility.As shown in Figure 8B, L-cysteine is a reducing biomass that can reduce oxTMB to TMB.Therefore, we could use the excellent oxidase-like activity of Van-Pt1/Pd1 NPs to establish a standard curve for the L-cysteine assay.The experimental system included Van-Pt1/Pd1 NPs, TMB and L-cysteine.The UV-vis spectrum of the solution was detected using a UV-vis spectrophotometer.As shown in Figure 8C, the calibration showed a good linear relationship with the absorbance value at 652 nm.The standard detection equation of the Van-Pt1/Pd1 NPs for L-cysteine was Y = 0.379 + 15.367 × CL-cysteine (R 2 = 0.997), while the corresponding detection range of the L-cysteine concentration was 6-100 µM, and the detection limit was 0.0703 µM.Table 2 shows a comparison of the detection ranges and detection limits of L-cysteine for different materials.The Van-Pt1/Pd1 NPs (6-100 µM) had a wider linear range than the other sensors, including the MoS2-Au@Pt (0.8-54.4 µM), SPB@Pt NPs (0.4-3.5 µM) and Au-Ag (0.075-2 µM).In addition, Van-Pt1/Pd1 NPs had a lower detection limit (0.07 µM) than other sensors, such as PdPt3-LNT NDs (3.10 µM), Cu@Au/Pt (4.00 µM), VS4 NPs (2.50 µM) and CuMnO2 NFs (11.26 µM).Therefore, the colorimetric method had a wide linear detection range and a low sensitivity detection limit.•OH, respectively.

L-cysteine Assay
Here, the cytotoxicity of the Van-Pt 1 /Pd 1 NPs and Van was explored using the MTT assay.As shown in Figure 8A, when the concentration of the Van-Pt 1 /Pd 1 NPs and Van was 200 µg/mL, the cell viability was maintained above 90%.This indicated that the Van-Pt 1 /Pd 1 NPs were non-cytotoxic, as compared to previously reported Pt NPs and Pd NPs [52].Therefore, the Van-Pt 1 /Pd 1 NPs synthesized using the bio-template method have good biocompatibility.As shown in Figure 8B, L-cysteine is a reducing biomass that can reduce oxTMB to TMB.Therefore, we could use the excellent oxidaselike activity of Van-Pt 1 /Pd 1 NPs to establish a standard curve for the L-cysteine assay.The experimental system included Van-Pt 1 /Pd 1 NPs, TMB and L-cysteine.The UV-vis spectrum of the solution was detected using a UV-vis spectrophotometer.As shown in Figure 8C, the calibration showed a good linear relationship with the absorbance value at 652 nm.The standard detection equation of the Van-Pt 1 /Pd 1 NPs for L-cysteine was Y = 0.379 + 15.367 × C L-cysteine (R 2 = 0.997), while the corresponding detection range of the L-cysteine concentration was 6-100 µM, and the detection limit was 0.0703 µM.Table 2 shows a comparison of the detection ranges and detection limits of L-cysteine for different materials.The Van-Pt 1 /Pd 1 NPs (6-100 µM) had a wider linear range than the other sensors, including the MoS 2 -Au@Pt (0.8-54.4 µM), SPB@Pt NPs (0.4-3.5 µM) and Au-Ag (0.075-2 µM).In addition, Van-Pt 1 /Pd 1 NPs had a lower detection limit (0.07 µM) than other sensors, such as PdPt 3 -LNT NDs (3.10 µM), Cu@Au/Pt (4.00 µM), VS 4 NPs (2.50 µM) and CuMnO 2 NFs (11.26 µM).Therefore, the colorimetric method had a wide linear detection range and a low sensitivity detection limit.The selectivity of the Van-Pt 1 /Pd 1 NPs was investigated by detecting L-cysteine (L-cys) and potentially interfering substances, such as Mg 2+ , alanine (Ala), phenylalanine (Phe), leucine (Leu), glycine (Gly), proline (Pro), glutamic acid (Glu), maltose (Mal), lactose (Lac) and fructose (Fru).As shown in Figure 8D, the absorbance of L-cysteine was much higher than that of the other interfering agents, even when the concentration of interfering agents was three times higher than that of L-cysteine.This indicated that the Van-Pt 1 /Pd 1 NPs had good selectivity as probes for detecting L-cysteine.The selectivity of the Van-Pt 1 /Pd 1 NPs for L-cysteine was high and reasonable, as compared with other reports [58,62].
-Pt 2 -Pd 1 NPs and Van-Pd 1 -Pt 2 NPs had a slight degree of aggregation.The Van-Pt 2 /Pd 1 NPs and Van-Pt 1 /Pd 1 NPs had better dispersion.The size of the Van-Pt m /Pd n NPs was very small.The aggregation of the Van-Pt 2 -Pd 1 NPs and Van-Pd 1 -Pt 2 NPs may be due to the long reaction time.

Figure 3 .
Figure 3. (A) Hydrodynamic size of Van-Pt m /Pd n NPs.(B) Zeta potential of Van-Pt m /Pd n NPs, (C) catalytic activities of Van-Pt m /Pd n NPs with different synthesis methods and (D) absorbance of (C) at 652 nm, (m = 1, 2; n = 1, 2), Van-Pt m /Pd n NPs reacted with TMB at 25 • C for 5 min.
• and 46.24 • are attributed to the planar crystal structure of Pt and Pd nanoparticles.Comparing the reference code 01-001-1194 for Pt and the reference pair 46-1043 for Pd, the diffraction peak is slightly higher than that of Pt and slightly lower than that of Pd.It is clear that the diffraction angles of the Van-Pt 1 /Pd 1 NP alloy are in the middle of the diffraction peaks of Pt and Pd [41].Thus, the XRD of the Pt-Pd alloy nanoparticles proved that we successfully synthesized Van-Pt 1 /Pd 1 NPs using Van.The other peaks observed at 67.45 • , 81.28 • and 85.71 • were related to the planar formation of Pt and Pd in the Van-Pt 1 /Pd 1 NPs.Biomolecules 2023, 13, x 9 of 18

Figure 5 .
Figure 5. (A) Oxidase-like activity of Van-Pt1/Pd1 NPs with a reaction time of 5 min at 25 °C.(B) Peroxidase-like activity of Van-Pt1/Pd1 NPs with a reaction time of 2 min at 25 °C.(C) Optimal pH and (D) optimal temperature of Van-Pt1/Pd1 NPs.

Figure 5 .
Figure 5. (A) Oxidase-like activity of Van-Pt 1 /Pd 1 NPs with a reaction time of 5 min at 25 • C. (B) Peroxidase-like activity of Van-Pt 1 /Pd 1 NPs with a reaction time of 2 min at 25 • C. (C) Optimal pH and (D) optimal temperature of Van-Pt 1 /Pd 1 NPs.We also designed different groups to investigate the peroxidase-like activity ofVan-Pt 1 /Pd 1 NPs: 1TMB + H 2 O 2 , 2Van-Pt 1 /Pd 1 NPs + H 2 O 2 , 3TMB + Van-Pt 1 /Pd 1 NPs and 4TMB + Van-Pt 1 /Pd 1 NPs + H 2 O 2 .As shown in Figure5B, the characteristic absorption peak of oxTMB at 652 nm was the highest in the Van-Pt 1 /Pd 1 NPs + TMB + H 2 O 2 group after 2 min of reaction.In addition, the TMB + H 2 O 2 and Van-Pt 1 /Pd 1 NPs + H 2 O 2 groups did not show the characteristic absorption peak of oxTMB at 652 nm.Importantly, oxTMB peaks were observed in the TMB + Van-Pt 1 /Pd 1 NP group under the influence of Van-Pt 1 /Pd 1 NPs oxidase-like activity, but the absorbance was 25% lower than that of the TMB + Van-Pt 1 /Pd 1 NPs + H 2 O 2 group.Therefore, the Van-Pt 1 /Pd 1 NPs had not only good oxidase-like activity but also peroxidase-like activity.The oxidase-like activity of the Van-Pt 1 /Pd 1 NPs was very high; thus, the oxidase-like activity of the Van-Pt 1 /Pd 1 NPs was investigated in the subsequent experiments.The oxidase-like activity of Van-Pt 1 /Pd 1 NPs is influenced by external conditions, the main influencing factors being pH and temperature.In order to find the optimal conditions for enzyme catalysis, the oxidase-like activity of the Van-Pt 1 /Pd 1 NPs at different pHs and temperatures was investigated.From Figure5C, it can be seen that the Van-Pt 1 /Pd 1 NPs had the best oxidase-like activity at pH = 3.The oxidase-like activity of the Van-Pt 1 /Pd 1 NPs decreased at other pHs.Therefore, the optimal pH for the oxidase-like activity of the Van-Pt 1 /Pd 1 NPs was 3. The pH affects the binding of the TMB with the Van-Pt 1 /Pd 1 NPs.The substrate TMB should not be suitable for binding to the Van-Pt 1 /Pd 1 NPs at a non-optimal pH, leading to a decrease in oxidase-like activity.Furthermore, Van-Pt 1 /Pd 1 NPs should have the highest amount of reactive oxygen species at pH = 3.The effect of temperature on the enzyme activity was then explored under the conditions of the optimal pH.In

Figure 6 .
Figure 6.(A) Catalytic kinetic diagram of different TMB concentrations.(B) is the double-inverse curve of (A).(C) The seven-day stability of the Van-Pt1/Pd1 NPs and (D) the stability of the Van-Pt1/Pd1 NPs at different temperatures.Van-Pt1/Pd1 NPs were incubated at different temperature for 2 h.

Figure 6 .
Figure 6.(A) Catalytic kinetic diagram of different TMB concentrations.(B) is the double-inverse curve of (A).(C) The seven-day stability of the Van-Pt 1 /Pd 1 NPs and (D) the stability of the Van-Pt 1 /Pd 1 NPs at different temperatures.Van-Pt 1 /Pd 1 NPs were incubated at different temperature for 2 h.

3. 3 .
Mechanism of the Oxidase-Like Activity of Van-Pt 1 /Pd 1 NPs The oxidation reaction of TMB based on Van-Pt 1 /Pd 1 NPs is closely related to reactive oxygen species [20].Reactive oxygen species include as singlet oxygen ( 1 O 2 ), hydroxyl radical (•OH), superoxide anion (O 2 • − ), etc.The mechanism of the oxidase-like activity of Van- Pt 1 /Pd 1 NPs was investigated by adding different reactive oxygen species inhibitors, such as BQ, NaN 3 and IPA, which have quenching effects on O 2 • − , 1 O 2 and •OH, respectively.

Figure 7 .
Figure 7. (A) Mechanism of oxidase-like activity and (B) catalytic process of Van-Pt 1 /Pd 1 NPs.The reaction was at 30 • C, pH = 3, for 5 min.BQ, NaN 3 and IPA had suppressive effects on O 2 − , 1 O 2 and

Table 1 .
Comparison of K m and V max .

Table 2 .
Comparison of L-cysteine detection range and detection limit for different materials.

Table 2 .
Comparison of L-cysteine detection range and detection limit for different materials.