2D PdCu Alloy Nanodendrites Manifest Effective Peroxidase-Like Activity Against Bio lms


 Noble metal nanomaterials with peroxidase-like catalytic activity have received great interest lately for their potential applications in biomedicine and environmental protection; however, it is still challenging to achieve high catalytic efficiency despite enormous efforts. In this work, a novel but simple route was developed to synthesize 2D PdCu alloy nanodendrites (PdCu NDs) as a high-performance peroxidase mimic for biofilm elimination. Catalytic kinetics shows that the composition-dependent synergy between Pd and Cu in the PdCu NDs can strongly enhance the peroxidase-like activity. Density functional theory calculations further provide the underlying mechanisms at both atomic and electronic levels for the effective adsorption and dissociation of H2O2 molecules on PdCu NDs surfaces. Owing to their superior peroxidase-like activity, the PdCu NDs exhibit striking biofilm inhibition properties, which suggests that the controlled synthesis of 2D noble metal alloy may open up new opportunities for enhancing enzyme-like activities of noble metal nanomaterials.


Introduction
Infections induced by bio lm-forming bacteria have emerged as a severe public health threat around the world due to their increasing resistance against different antibiotics. [1][2][3] The bacteria in the bio lm state have increased resistance to antibiotics, disinfectants, heat stress, pH, and immunity, which is one of the main reasons for the di culties in the treatment of bio lm infection. 4 For example, the opportunistic pathogen Pseudomonas aeruginosa (P. aeruginosa) is one of the leading causes of nosocomial infection, 5 because the formed bio lm makes it extremely di cult to eradicate P. aeruginosa thoroughly. 6 Therefore, it is of urgent need to develop highly e cient antibacterial agents with unique inhibition mechanism to defeat the bio lm without inducing signi cant resistance.
Meanwhile, a wide variety of nanomaterials have been proposed/developed as promising antibacterial agents, such as silver nanomaterials, 7 copper nanomaterials, 8 and graphene oxide, 9 owing to the rapid development of nanotechnology. However, these nanomaterials have achieved limited success in antibio lms, primarily due to their not-high-enough catalytic activities. In recent years, nanomaterials with intrinsic enzyme-like catalytic activities (also known as nanozymes) have attracted great attention because of their high catalytic activities and high stability (and low cost). [10][11][12] Given these advantages, nanozymes have shown great potential in a broad range of applications including biological detection, 13,14 cancer treatment, 15,16 and antibacterial agent. 17,18 Previous studies have demonstrated that Pd nanozymes are good candidates for antibacterial application because of their high catalytic activity and good biocompatibility. 19,20 For instance, our group found in an earlier study that Pd nanomaterials with peroxidase-like activity exhibit excellent antibacterial effects without producing signi cant toxicity to human cells, which can be used as new antibacterial agents. 21 Nonetheless, there is still much work to do to improve the catalytic activity of Pd nanomaterials as antibacterial agents, particularly for anti-bio lms. To the best of our knowledge, few reports have provided 2D Pd-based nanomaterials with enzyme-like catalytic activity for bio lm elimination so far.
In order to further improve the catalytic activity of Pd nanomaterials, one feasible strategy is to tune their composition 22 and structure 23 . Many reports have revealed that the enzyme-like activities of Pd nanomaterials can be enhanced by a combination of Pd with other metals (eg. Au, 24 Pt, 25 Ir 26 ) to form alloy nanostructures. For instance, depositing Ir atoms as ultrathin shells on Pd nanocubes signi cantly enhanced the peroxidase-like e ciency compared with the original Pd nanocubes. 26 Moreover, it was demonstrated that two-dimensional (2D) Pd-based nanomaterials display superior catalytic activities over commercial Pd black catalysts under similar conditions, due to the high surface-area-to-volume ratio and thus high density of exposed atoms on the surface of 2D nanomaterials. 27,28 Very recently, Zhang and coworkers fabricated PdCu alloy nanosheets, which exhibit much higher electrocatalytic activity than those of Pd-based catalysts in formic acid oxidation. 29 However, despite PdCu alloy nanosheets' good catalytic activity, their synthetic process was complicated. They were synthesized in the oil phase, which makes them di cult to be directly applied in biomedical applications. Meanwhile, 2D alloy nanodendrites with high structural anisotropy and speci c surface areas possess extensive undercoordinated sites, which can supply a natural dendrite-like framework for the study of defect engineering. 30 However, the stringency of their preparing process, originated from the thermodynamically unfavorable (and mostly kinetical driven) rami cation process within the 2D, suppress somewhat the promise for catalytic applications. 31 As a result, a better and more straightforward preparation of 2D dendrite-like alloy is highly desired, though very challenging.
In this study, we developed a novel but simple method to synthesize 2D PdCu alloy nanomaterials with dendrite-like morphology (PdCu NDs) in aqueous solution under mild conditions for bio lm elimination.
Our experiments show that PdCu NDs mimic peroxidase and the peroxidase-like activity of PdCu NDs can be effectively regulated by varying Pd/Cu ratio. Density functional theory (DFT) calculations reveal atomic and electronic details on how Pd/Cu ratio affects the catalytic property of PdCu NDs. Furthermore, we show that PdCu NDs with intrinsic peroxidase-like acticity exert a strong anti-bio lm acticity.

Preparation and characterization of the PdCu NDs
The PdCu NDs were synthesized by the coreduction of Pd and Cu precursors in aqueous solution with the presence of octadecyltrimethylammonium chloride (OTAC) at 10 o C (see Supporting information for more details). This novel method has two advantages: (1) the reaction condition is mild and environmentally friendly; (2) the products can be easily dispersed in water, which is favorable for biomedical applications.
The crystal structure of the obtained PdCu NDs was analyzed rstly by X-ray powder diffraction (XRD). As shown in Fig. 1a, XRD pattern supports the formation of PdCu alloy structures. The diffraction peaks of the products were between the corresponding peaks of pure Pd and Cu, indicating the formation of PdCu alloy structures rather than phase separation. 31 According to the transmission electron microscopy (TEM) images, the lateral size of the as-synthesized PdCu NDs are around 50 nm and a dendrite-like structure with an obvious center and branched margins were observed ( Fig. 1b and c). The high resolution TEM (HRTEM) image and selected area electron diffraction (SAED) pattern of PdCu NDs exhibited clear lattice pattern, where a typical lattice spacing of 2.1 Å was observed in accordance with that of lattice spacing of PdCu alloy (111 facet), suggesting a certain well-de ned crystal structure of PdCu NDs (Fig. 1d).
Moreover, high-angle annular dark-eld scanning TEM (HAADF-STEM) together with energy dispersive Xray spectroscopy mapping (EDX) disclosed that Pd (red) and Cu (yellow) distribute homogeneously in the nanocrystals, con rming the successful formation of PdCu NDs (Fig. 1e). The thickness of the PdCu NDs was measured by atomic force microscopy (AFM). From a random height pro le across the nanocrystals, we found that PdCu NDs exhibited 2D structure with an average thickness of ≈ 7 nm (Fig. 1f).
Next, the molar ratio of Pd and Cu precursors was further varied to explore and optimize the catalytic activity of the synthesized PdCu NDs. It was found that pure H 2 PdCl 4 formed Pd nanoparticles composed of two-dimensional slices (Fig. 2a). The typical 2D PdCu NDs were obtained when the molar ratio of Pd to Cu precursors increased to 20:6 ( Fig. 2c). Additionally, when the molar ratio of Pd to Cu precursors increased from 20:6 to 20:15, the nanodendrite-like PdCu nanostructures had no signi cant change •OH and then oxidize the TMB. As shown in Fig. 3a & Fig. S1, Pd nanoparticles show good peroxidase-like activity for oxidation of TMB, as evidenced by the time-dependent increment in the maximum absorbance (652 nm). However, after the introduction of Cu, even at relatively low Cu content, the oxidizing ability of TMB was signi cantly enhanced. It was found that the Pd 9.2 Cu NDs exhibit the most effective oxidizing ability of TMB in this case.
We subsequently carried out a steady-state kinetic analysis to quantify the catalytic e ciency of PdCu nanostructures. Typical Michaelis−Menten kinetics were observed within the suitable range of H 2 O 2 concentration (Fig. 3b) and a series of kinetic parameters were summarized in Table S1. It can be seen that the Michaelis constant (K m ) values of PdCu nanostructures followed a gradual downward trend as the Cu contents increasing in the nanostructures (Fig. 3c). This indicates that the adsorption a nity of PdCu nanostructures with H 2 O 2 can be enhanced by increasing the Cu content. However, the k cat value, which measures catalytic e ciency of the catalyst, 32 showed a volcano-shaped dependence on the Cu contents, with maximum points corresponding to Pd 9.2 Cu NDs, which is 9-fold higher than that of Pd 7 Cu NDs. As a peroxidase-like nanozyme, Pd 9.2 Cu NDs could catalyze terephthalic acid (TA) into highly uorescence 2-hydroxy TA (TAOH) in the presence of H 2 O 2 , indicating the formation of •OH (Fig. 3d).
Moreover, we found that the catalytic ability of the Pd 9.2 Cu NDs was highly dependent on the pH and temperature of the reaction system (Fig. S2).

Quantum mechanics calculations on the catalytic activity
To gain more insight into the catalytic mechanism of PdCu NDs, we then performed density function theory (DFT) calculations to study the adsorption of H 2 O 2 on PdCu NDs surfaces. As shown in Fig. 4a- showed that substantial charge transfers occurred from Cu to Pd atoms, in agreement with previous calculations and experimental measurements (Fig. S4). 33,34 Also, as the Cu content increases, the electron transfer between Pd and Cu becomes more profound in a linear way, leading to greater electrostatic interactions between H 2 O 2 and PdCu NDs surface. Fig. 4g shows a typical EP distribution of the H 2 O 2 -metal interface: the EP is positive in the region between H 2 O 2 and the metal surface (region a) and negative in the region away from H 2 O 2 and the metal surface along the z-axis (region b). By separately scanning the EPs curves of Pd 11.5 Cu, Pd 6.7 Cu and H 2 O 2 (Fig. S5a), we found that in the region a, the EPs of the isolated H 2 O 2 and the bimetallic slab are positive (Fig. S5b), and the EP of Pd 11.5 Cu is higher than that of Pd 6.7 Cu, indicating a greater repulsion of Pd 11.5 Cu to H 2 O 2 . In region b, however, it seems the opposite, i.e. EP (Pd 11.5 Cu) < EP (Pd 6.7 Cu), but the EP of H 2 O 2 is negative. Therefore, Pd 6.7 Cu provides a greater attraction in region b to the adsorbed H 2 O 2 . Taken together, both regions demonstrate a more favorable electrostatic adsorption of the H 2 O 2 molecule on Pd 6.7 Cu than Pd 11.5 Cu (i.e., the higher the Cu content, the stronger the adsorption).
The peroxidase-like activity on the metal surface in this study involves the two-step process as below: According to the previous studies, the homolytic cleavage of H 2 O 2 (Eq. 1) is the rate-determining step, 35,36 and the overall reaction energy (E r ) can be employed to indicate the peroxidase-like activity: a more negative value of E r implies a higher peroxidase-like activity. In the lowest-energy adsorption structures for the cleaved H 2 O 2 , the produced •OH preferred to be adsorbed at the bridge site between Pd and Cu (Fig. S6). Furthermore, the dissociation (or more precisely, the dissociative chemisorption) of H 2 O 2 (Eq. 2) showed the most negative E r value (−39.01 kcal mol −1 ) on Pd 8

Anti-bio lm activity of the PdCu NDs
Our experimental and theoretical results demonstrate that PdCu NDs exhibit outstanding peroxidase-like activity by e ciently generating H 2 O 2 species that are subsequently converted into •OH radicals. The •OH radicals are highly reactive species that attack most of the organic molecules. They are highly oxidasive in nature which is attributed to their strong catalytic potential. It has been reported that nanozymes with peroxidase-like activity convert H 2 O 2 into •OH radicals, which are more toxic to bacteria. 18 As PdCu NDs induce the signi cant formation of •OH radicals, we then examined whether PdCu NDs mediated H 2 O 2 catalysis can eliminate the embedded bacteria in bio lms. Bio lms were formed using P. aeruginosa, a well-established bio lm-forming pathogen. We rst investigated the effects of Pd 9.2 Cu NDs (and/or H 2 O 2 ) on the integrity of bio lms. The extracellular polymeric matrix and bacterial cells were labelled with an Alexa Fluor 647-dextran (in red) and SYTO 9 (in green), respectively. After 72 h growth, the bacterial cells are densely packed with an extracellular polymeric matrix forming a 3D bacterial structure (bio lm) in the untreated control group (Fig. 5a). Confocal microscopy imaging revealed that treatments with Pd 9.2 Cu NDs and/or H 2 O 2 impaired both the accumulation of bacterial cells and the development of extracellular polymeric matrix. According to quantitative image analysis, the thickness of the bio lms decreased from 39 ± 7 µm to around 30 µm when treated with Pd 9.2 Cu NDs or H 2 O 2 alone; the thickness further decreased to 10 ± 2 µm when treated with both Pd 9.2 Cu NDs and H 2 O 2 (Pd 9.2 Cu NDs/H 2 O 2; i.e., H 2 O 2 were treated immediately after the Pd 9.2 Cu NDs treatment; see Fig. 5b). Also, bio lms were quantitatively evaluated by counting the number of viable bacterial cells (Fig. 5c). Bio lms treated with Pd 9.2 Cu NDs/H 2 O 2 exhibit an exceptionally strong biocidal effect against P. aeruginosa. In contrast, treatments with Pd 9.2 Cu NDs or H 2 O 2 alone had limited antibacterial effects. All these results suggest that Pd 9.2 Cu NDs/H 2 O 2 is a suitable agent for eliminating bio lms.

Conclusion
In summary, with a novel but simple synthesis method, 2D PdCu NDs have been successfully prepared via the coreduction of Pd and Cu precursors in aqueous solution. PdCu NDs demonstrate enhanced peroxidase-like activity compared to that of monometallic Pd nanomaterials. In particular, the peroxidaselike activities of PdCu NDs are further improved by tuning the molar ratio of Pd/Cu, as the Cu content regulates the surface d-electrons in a non-linear manner. The distinct peroxidase-like properties of the ne-tuned PdCu NDs endow them with excellent bio lm elimination capability via the generation of hydroxyl radicals. Our work offers great opportunity to design noble metal nanozymes with enhanced performance, which might advance the development of nanozymes as a new class of highly e cient antibacterial agents. Declarations ratios. Figure. S5. (a) The EP of Pd 11.5 Cu slab model (white, H; red, O; cyan, Pd). (b) The separately scanned EP of Pd 11.5 Cu, Pd 6.7 Cu and H 2 O 2 along the path denoted in panel (a). Figure S6. The lowestenergy adsorption structures for the coadsorption of two •OH on PdCu bimetallic surface on Pd 11.5 Cu.

Supplementary Files
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