Enhanced Peroxidase-Like and Antibacterial Activity of Ir-CoatedPd-Pt Nanodendrites as Nanozyme

To inhibit the growth of bacteria, the DA-PPI nanozyme with enhanced peroxidase-like activity was synthesized. The DA-PPI nanozyme was obtained by depositing high-affinity element iridium (Ir) on the surface of Pd-Pt dendritic structures. The morphology and composition of DA-PPI nanozyme were characterized using SEM, TEM, and XPS. The kinetic results showed that the DA-PPI nanozyme possessed a higher peroxidase-like activity than that of Pd-Pt dendritic structures. The PL, ESR, and DFT were employed to explain the high peroxidase activity. As a proof of concept, the DA-PPI nanozyme could effectively inhibit E. coli (G−) and S. aureus (G+) due to its high peroxidase-like activity. The study provides a new idea for the design of high active nanozymes and their application in the field of antibacterial.


Introduction
Over the last two decades, the increasing spread of bacterial infections in medical community led to high mortality and morbidity and became a serious problem worldwide [1]. Antibiotics were the most widely used drugs to treat the bacterial infections and saved countless human lives. However, the overuse of antibiotics leads the bacteria such as Staphylococcus aureus (G − ) and Escherichia coli (G + ) to be multidrug-resistant, which turned out to be a major threat to hospitalised or immunocompromised patients [2]. Terefore, the new efective and safe drugs to eradicate bacterial infections need to be developed.
Enzyme is an indispensable core substance of life. Many key technologies and product manufacturing, such as in bionics [3], environmental treatment [4], antibacterial [5], and biomedical applications [6], are inseparable from enzymes. However, the applications in these areas of enzymes are limited by the low stability, time-consuming preparation, and purifcation. Nanozymes are inorganic nanomaterials that possess the enzyme-like activity (such as peroxidase-like activity). Compared to the nature enzyme, nanozymes possess the advantage of easy preparation, adjustable activity, and high stability. Tus, nanozymes have the potential to replace biological enzymes. Te peroxidase-like activity is widely used in felds of biomarkers detection [7], antitumor [8], antibacterial, and antiviral [9]. However, the low activity has become a bottleneck limiting the wide application of nanozymes.
After decades of development, signifcant progress has been made in the activity optimization of nanozymes. Jiang et al. [10] found that the smaller the particle size, the higher activity of the Fe 3 O 4 . However, the stability of the nanozymes will decrease while the particle size is reduced to a certain size (<2 nm). Nowadays, the structures with high specifc surface area (porous and dendrites) have been applied to enhance the activity of nanozymes. For example, Chen et al. [11] found that porous materials can enhance the peroxidase-like activity of nanozymes. Te Pt-Pd porous nanorods showed a higher peroxidase-like activity than that of Pd-Pt nanorods and Pd-Pt nanoparticles [12]. In contrast, dendritic structures have attracted extensive attention due to the large specifc surface area, high mass transfer efciency, and a large number of low-coordination atoms. Ge [13] found that branched structures with high density of active site can enhance the activity of Pt hollow nanodendrites.
In the previous reports, the branch ends of the dendritic structure are mostly monometallic. Te peroxidase-like activity of monometallic nanozyme is always weaker than that of multimetallic nanozyme [14,15]. Te Sabatier principle states that the catalyst-intermediate interaction should be moderate [16,17]. Te alloying is an appropriate way to adjust the interaction between the surface of catalyst and intermediate. For example, the Au doped Pd twodimensional nanosheet showed a higher peroxidase-like activity compared to Pd nanosheet [18]. Among the transition metal, thanks to the high afnity to H 2 O 2 , iridium (Ir) is always used to form alloy to enhance the peroxidase-like activity of nanozymes [19]. However, the iridium is rarely applied to form alloy on nanodendrites nanozymes.
Here, the Ir was used to coat on the branch ends of Pd-Pt nanodendrites (named D-PP nanozyme) to form alloyed Pd-Pt-Ir nanozyme (named DA-PPI nanozyme). Te DA-PPI nanozyme shows a higher peroxidase-like activity than that of D-PP nanozyme. Te DA-PPI nanozyme was fnally applied in the inhibition of E. coli (G − ) and S. aureus (G + ).

Te Preparation of D-PP Nanozyme.
Te solution (5 mL) containing diferent amount of K 2 PdBr 4 and K 2 PtCl 6 was frstly prepared. Pluronic F127 (0.05 g) was then dissolved into the above solution. Te reducing agent (ascorbic acid, 0.1 M, 5 mL) was injected into the above mixture. Te mixture was incubated at 30°C for 12 h. During the synthesis process of Pd-Pt nanodendrites (D-PP nanozyme), the mixture turned from brown to black. Finally, the D-PP nanozyme was obtained through centrifugal washing and stored in 10 mL ethylene glycol.

Te Preparation of DA-PPI Nanozyme.
First, about 1 mL D-PP nanozyme solution was injected into a fask with 9 mL EG. Ten, the ascorbic acid (100 mg) and PVP (60 mg) were added into the mixture. Te mixture was heated up to 110°C and incubated for 30 min. After the incubation, the mixture was heated up to 180°C. Te Na 3 IrCl 6 EG solution (1.5 mL, 0.15 mg ml −1 ) was slowly injected into the mixture. After the injection, the mixture was cooled down to the room temperature. Te DA-PPI nanozyme was obtained through centrifugal washing with acetone once and deionized water three times. Te obtained DA-PPI nanozyme was stored in 1 mL deionized water for next use. Te concentration of DA-PPI nanozyme was detected to be 18.6 mgL −1 (detected as Pd) using ICP-MS. Te preparation of DA-PPI can be summarized as the following chemical equation: (1) Te yield (y) was calculated as the following equation: Tus, the y is calculated to be 3.5% according to the equations (1)-(3).

Peroxidase-Like Activity and Kinetic Assay of DA-PPI
Nanozyme. Te peroxidase substrate (TMB) can be oxidised by H 2 O 2 with the catalyst of HRP. Te nanozymes possess the similar activity to HRP (peroxidase-like activity). Tus, the peroxidase-like activity of DA-PPI nanozyme is assessed by the catalytic oxidation of the TMB in the presence of H 2 O 2 . Te oxidation product of TMB has an obvious UV absorption peak at 652 nm. Te molar extinction coefcient (ε) is 3.9 × 10 4 M −1 cm −1 . Te bufer is acetate bufer with pH value of 4.0. Te amount of DA-PPI nanozyme used in this part is 10 μL (18.6 mg/L). During the measurement of enzyme kinetic parameters of DA-PPI nanozyme towards H 2 O 2 , the concentration of TMB is fxed at 20.80 mM. Te concentration of H 2 O 2 ranged from 1.04 to 33.33 mM. Te absorbance at 652 nm of diferent groups was recorded in 3 minutes. Similarly, when measuring the enzyme kinetic parameters of DA-PPI nanozyme towards TMB, the concentration of H 2 O 2 was fxed at 62.5 mM. Te concentration of TMB ranged from 0.10 to 6.67 mM. Te parameters such as K m and V max are calculated by the following equations: where [s] is the substrate concentration, v is the initial reaction rate, ΔA is the change of absorbance, Δt is the change of time, ε is the absorbance coefcient of the substrate oxidized TMB, which is usually 39000 M −1 cm −1 at 652 nm, and l is the path length of light through the colorimetric dish (cm).

Result and Discussion
3.1. Te Characterization of DA-PPI Nanozyme. In this part, the morphology of DA-PPI nanozyme was characterized by a scanning electron microscope (SEM). In Figure 1(a), the DA-PPI nanozyme is nearly spherical with a uniform morphology. Subsequently, the morphology of the DA-PPI nanozyme was characterized by transmission electron microscopy (TEM). Te DA-PPI nanozyme had an urchin-like structure with a large number of gaps at the branch ends providing a guarantee for the high catalytic activity (Figure 1(b)). In order to confrm the composition of DA-PPI nanozyme, the element distribution was characterized by Xray spectroscopy (EDX). Te results present that the Pd and Pt are distributed in the core and shell, respectively. Te Ir is distributed on the surface of DA-PPI nanozyme. Te results indicate that the elements in DA-PPI nanozyme are distributed similar to a sandwich. An alloy composed by Ir and Pt may form on the surface of DA-PPI nanozyme.
According to the EDX result, the DA-PPI nanozyme is composed by Pd, Pt, and Ir. However, the combination style of these three elements is not clear. Tus, the X-ray photoelectron spectroscopy (XPS) was employed to characterize the valence state changes of each element in D-PP nanozyme and DA-PPI nanozyme. Te corresponding results are shown in the fgure (Figure 2). In Figure 2(a), it can be seen that D-PP nanozyme contains not only Pd and Pt but also exogenous oxygen and carbon elements. Similar to D-PP nanozyme, DA-PPI nanozyme contains several elements of Pd, Pt, O, and C. What is more, the Ir can be observed in DA-PPI nanozyme according to the XPS broad spectrum. In the narrow spectrum of Pt-Pt, the Pt is split to Pt4f 5/2 and Pt4f 7/2 . Te corresponding peaks are located at 74.66 eV and 71.35 eV, respectively. In contrast, the Pt4f 5/2 and Pt4f 7/2 in DA-PPI nanozyme are located at 74.80 eV and 71.47 eV, respectively. After the deposition of Ir on the surface of D-PP nanozyme, the binding energy of Pt shifts to high felds. Te results indicate that the chemical bond between platinum and iridium was formed during the deposition. Te result is consistent with the previous report [20]. Furthermore, the narrow spectrum of Ir in DA-PPI nanozyme was analyzed. Te results present that the Ir element contains Ir 0 and Ir δ+ (Figure 2(c)). Te Ir 0 is split to Ir 0 4f 5/2 and Ir 0 4f 7/2 . Te corresponding peaks are located at 63.70 eV and 60.74 eV, respectively. Te splitting peaks of Ir δ+ (namely, Ir δ+ 4f 5/2 and Ir δ+ 4f 7/2 ) are located at 64.48 eV and 61.55 eV, respectively. Tis result is consistent with the previous report about the Ir characteristic peak [21].

Te Peroxidase-Like Activity Assessment of DA-PPI
Nanozyme. DA-PPI nanozyme can catalyze the oxidation of TMB with the participation of hydrogen peroxide (Figure 3(a)). Te oxidation product is blue with obvious UV absorption at 652 nm (Figure 3(b)). Te intensity refects the peroxidase-like activity of the nanozyme. During the catalytic process, the absorbance at 652 nm increased with the increase of time. By adjusting the concentration of DA-PPI nanozyme (0.4-6.0 μg/L), the absorbance at 652 nm increased with the increase of concentration in 3 min (Figure 3(c)). To evaluate its stability, the DA-PPI nanozyme was applied for catalyzing the oxidation of TMB for fve cycles. Te result shows that the intensity of absorbance at 652 nm remained basically unchanged for fve cycles indicating the DA-PPI nanozyme possess an eligible stability (Figure 3(d)).
Te catalytic process of the nanozyme ft for the Michaelis-Menton kinetics [22]. Te substrates include hydrogen peroxide and TMB. In order to measure the kinetic parameters of the DA-PPI nanozyme towards H 2 O 2 , the concentration of TMB is fxed frst. Te concentration of H 2 O 2 is varied to detect and calculate the parameters of K m and V max . Te results are shown in Figure 4(a). As the concentration of H 2 O 2 increases, the maximum reaction rate gradually approaches equilibrium. Combined with the double-reciprocal curve (Figure 4(b)), the relevant parameters of the catalytic efciency of nanozymes for hydrogen peroxide are calculated and obtained. Te results show that the afnity of DA-PPI nanozyme for hydrogen peroxide was better than that of D-PP nanozyme (4.51 mM vs. 9.28 mM, Table 1), but slightly less than that of graphene quantum dots (2.288 mM) and graphene oxide (2.301 mM) [23]. Similarly, the corresponding parameters of TMB were measured. Te results are shown in Figures 4(c) and 4(d). Te afnity of DA-PPI nanozyme for TMB was close to that of D-PP nanozyme (0.10 mM vs. 0.16 mM, Table 1). Tese results suggest that DA-PPI nanozyme exhibits a relative higher peroxidase-like activity than that of D-PP nanozyme.

Te Intensity Assessment of Reactive Oxygen Species.
In order to explain the high activity of DA-PPI nanozyme, terephthalic acid (TA) was employed to capture the hydroxyl radical in the system (photoluminescence (PL) method). Te reaction product of TA and hydroxyl radical shows an obvious fuorescence emission at 450 nm [24]. Te fuorescence intensity represents the amount of hydroxyl radicals in the system containing nanozyme and H 2 O 2 . In this part, 10 μL of Pd-Pt-Ru solution was added to 2.5 mL of acetic acid bufer solution (HAC-NaAC pH � 4) containing 100 μL of TA (0.05 mM) and 400 μL H 2 O 2 (62.50 mM). Te fuorescence spectra of the samples were collected after 30 minutes of reaction by fuorescence spectroscopy (F-7100FL). Te result is shown in Figure 5(a). It can be seen that the amount of hydroxyl radicals generated in the DA-PPI nanozyme group is signifcantly higher than that of the D-PP nanozyme group. Furthermore, the ESR was used to confrm the amount of hydroxyl radicals generated in the system of nanozyme and H 2 O 2 . Te DMPO (5,5-dimethyl-1-pyrroline N-oxide) was employed to capture the hydroxyl radicals in the DA-PPI nanozyme and D-PP nanozyme group. Te reaction product of DMPO and hydroxyl radical will produce a characteristic splitting peak of 1 : 2 : 2 : 1 [25]. Te result is shown in Figure 5(b). Te intensity of the splitting peak represents the amount of hydroxyl radicals in the system. It can be seen that the amount of hydroxyl radicals generated by the DA-PPI nanozyme group is signifcantly higher than that of the D-PP nanozyme group. Te result is consistent with the PL methods. Te high amount of hydroxyl radicals generated by the DA-PPI nanozyme group lead up to the high peroxidase-like activity.
Te PL and ESR spectra present that DA-PPI nanozyme can produce a relative high amount hydroxyl radical with H 2 O 2 participated. However, the reason is not clear. In this part, the density functional theory (DFT) was employed to analyze the adsorption energy of D-PP nanozyme and DA-PPI nanozyme to H 2 O 2 and OH [26][27][28].   (Figure 6(d)), which coincides to the optimal value of previous report [29]. Te results indicate that the appropriate adsorption energy of OH on Ir (111) endows DA-PPI nanozyme a relative high peroxidase-like activity.

Te Antimicrobial Activity of DA-PPI Nanozyme.
It has been reported that some highly efcient nanozymes are capable of producing highly oxidative •OH by decomposing biosafety levels of H 2 O 2 to defend bacterial infections without toxicity to healthy tissues. For example, the oxygenated-group-enriched carbon nanotubes and Au/ g-C 3 N 4 hybrid nanozymes exhibit excellent peroxidase activity to produce reactive oxygen species (ROS) and act as efcient antibacterial agents for actual wound disinfection with low concentration of H 2 O 2 [30][31][32]. Te cytotoxicity test was carried out in this part. Te result shows that the DA-PPI nanozyme has minimal toxicity to human cells (Figure 7(b)). Te antibacterial activities of DA-PPI nanozyme against E. coli and S. aureus were then evaluated by measuring the survival ratios in our study. As presented in Figures 7(c)       concentration of H 2 O 2 will injure the normal tissues. Terefore, DA-PPI nanozyme could enhance the antibacterial activity of H 2 O 2 due to its high peroxidase-like activity (Figure 7(a)).

Conclusion
In this work, Ir was employed to deposit on the surface of Pd-Pd nanodendritic to form DA-PPI nanozyme. Te DA-PPI nanozyme was characterized by SEM, TEM, TEM-EDX, and XPS. Te DA-PPI nanozyme possessed a higher peroxidase-like activity compared to D-PP nanozyme. Te K m of DA-PPI nanozyme to H 2 O 2 is 2.06 times than that of D-PP nanozyme. According to the DFT calculation, the high peroxidase-like activity is attributed to the appropriate adsorption of Ir (111) to OH. Finally, the obtained DA-PPI nanozyme inhibits the growth of E. coli and S. aureus through peroxidase-like activity, which shows promise for overcoming the critical challenges in the treatment of bacterial infection (Scheme 1).

Data Availability
Te data used to support the fndings of this study are included within the article and are available from the corresponding authors upon request.

Conflicts of Interest
Te authors declare that they have no conficts of interest.  Bioinorganic Chemistry and Applications 9