A small library of copper-based metallenes with superior antibacterial activity

Abstract

We report the preparation of a small library of copper-based metallenes, such as copperene, brassene, bronzene, cupronickelene and AlCuZn trimetallene, via a cryo-pretreatment assisted liquid phase exfoliation method. To the best of our knowledge, these nanosheets may represent a new category of metallenes. Benefiting from mixed-valence copper-induced oxidative stress and cleavage effects of layered structures, the obtained metallenes could efficiently eliminate drug-resistant bacteria even at a concentration as low as 1 μg mL⁻¹. Due to the alloy engineering-induced change in the release rate of metal ions, the CuZn metallene exhibited a much better antibacterial ability than the other metallenes and three clinical antibiotics. We believe this work not only expands the category of emerging 2D metallenes, but also proposes a strategy combining 2D and alloy engineering to improve the antibacterial properties of copper-based materials.

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New concepts

As one of the oldest metallic antimicrobial agents, copper suffers from physiological toxicity and limited antibacterial performance, which critically hinder its antibacterial applications in clinics. Herein, we report the preparation of a small library of copper-based metallenes with superior antibacterial activity via a cryo-pretreatment assisted liquid phase exfoliation method. By converting conventional 3D copper-based particles into ultrathin 2D nanosheets through liquid exfoliation-induced dimensional engineering, five copper-based metallenes, including copperene, brassene, bronzene, cupronickelene and AlCuZn trimetallene (i.e., the two-dimensional topographies of elemental copper, CuZn, CuSn, CuNi and AlCuZn), were successfully prepared. Benefiting from their Fenton-like activity and ultrathin structure, the obtained metallenes could efficiently eliminate drug-resistant bacteria even at a concentration as low as 1 μg mL⁻¹, much better than pristine copper-based powders. Mechanistic studies revealed that the antibacterial effects were mainly attributed to mixed-valence copper-induced oxidative stress and cleavage effects of layered structures. Furthermore, the alloying effect-induced change in the release rate of metal ions could control the antibacterial capability of copper-based metallenes. When used for the treatment of MRSA-infected wound mice, metallenes exhibited a better wound healing effect than three conventional antibiotics. This work expands the category of emerging 2D metallenes and proposes a strategy fusing dimensional and alloy engineering to improve the antibacterial properties of copper-based materials.

Introduction

Metallenes, the two-dimensional (2D) topographies of metals, have emerged as rising stars in the fast-growing 2D materials family. As a new category of 2D materials proposed in recent years, metallenes are generally defined as atomically thin layers composed primarily of metallic atoms.^1,2 Compared to traditional metal nanoparticles, metallenes exhibit many exciting merits, including high atom economy, large specific surface area, high conductivity, and rich active sites,^3,4 thus demonstrating adaptability and functionality for a wealth of intriguing applications, such as energy storage and conversion,^5–7 electronics,^8–10 sensors,¹¹ and nanomedicine.^12–14 To date, a series of metallenes have been prepared via different methods.^15,16 Solid-melt exfoliation was used to prepare few-layer gallenene consisting of pure Ga atoms, which formed heterostructures with layered MoS₂.¹⁷ Recently, a one-pot wet-chemical approach was utilized to prepare an ultrathin PdMo bimetallene, which showed amazing catalytic activity.¹⁸ Compared with monometallenes, the alloying effects may lead to unexpected performance of metallenes, and thus the design and synthesis of alloy metallenes have drawn great interest.¹⁹ Nevertheless, stable and readily available alloy metallenes with superior activity are still rare.

Copper (Cu), one of the earliest metals utilized by human beings, plays a vital role in the development of early human civilizations.²⁰ The applications of copper and its alloys span various aspects of daily life, from vessels in the bronze age to electronics in modern society, and their antimicrobial properties have received special attention.^21,22 Compared to conventional antibiotics that raised a global challenge of bacterial resistance, copper is a low-cost antimicrobial metal with unique antibacterial mechanisms involving bacterial content leakage by breaking the outer cell membrane, disrupting the osmotic balance, and producing reactive oxygen species (ROS), which result in bacterial oxidation and DNA degradation.^23–25 Qian et al. utilized the strong coordination ability of copper ions with cellulose molecules to construct a wearable and washable antimicrobial cotton textile, which could effectively suppress various viruses and pathogenic bacteria in plants and animals.²⁶ Despite being widely used in daily life, copper-based materials always suffered from limited antibacterial capability, which was mainly associated with the copper valence state, contact surface area and release speed.²⁷ Copper alloying can promote the release of copper ions necessary for antibacterial efficacy. The amount of Cu released from a CuSn alloy is similar to that of commercial pure Cu.^28,29 This indicates that copper-based alloys can enhance their antibacterial properties by adding secondary metal elements. In addition, pure metal nanomaterials exhibit higher atomic utilization efficiency and abundant active sites and can also serve as nanozymes to play an excellent catalytic role in wound healing.^30–32 All these indicate that copper-based materials are promising antibacterial agents but their antibacterial activity needed to be further improved.

Herein, inspired by ancient bronze ware, we report a small library of metallenes containing five kinds of copper and copper alloy nanosheets with superior antibacterial capabilities (Fig. 1). A cryo-pretreatment assisted-liquid phase exfoliation method was used to prepare the 2D topographies of copper (Cu), brass (CuZn), bronze (CuSn), cupronickel (CuNi), and Devarda's alloy (AlCuZn), which were named copperene, brassene, bronzene, cupronickelene and AlCuZn trimetallene, respectively. By means of dimensional and alloy engineering strategies, the antibacterial activity of copper was greatly enhanced, and methicillin-resistant Staphylococcus aureus (MRSA) could be eliminated even at a concentration as low as 1 μg mL⁻¹. These antibacterial effects were mainly ascribed to mixed-valence copper-induced oxidative stress and cleavage effects of layered structures. In a MRSA-infected wound mice model, the CuZn metallene exhibited a better antibacterial ability than the other metallenes and three clinical antibiotics due to the alloying effect-induced change in the release rate of metal ions, thus accelerating the wound healing within 8 days. This work constructs a copper-based metallene library that is expected to fight against drug-resistant bacteria.

Fig. 1 Schematic illustration of the synthesis of copper-based metallenes for bacteria-infected wound healing.

Results

Synthesis of copper-based metallenes

Five kinds of metallic nanosheets, including copper (Cu), brass (CuZn), bronze (CuSn), cupronickel (CuNi), and Devarda's alloy (AlCuZn), were prepared using a combination of liquid nitrogen cryo-pretreatment and liquid-phase exfoliation in isopropyl alcohol (IPA). Low-temperature pretreatment in liquid nitrogen is used to tune the mechanical properties of the raw metal powders and is expected to enhance the exfoliation efficiency of copper and copper-based alloys and increase the yield of copper-based metallenes during subsequent ultrasonic treatment.^33,34 Scanning electron microscopy (SEM) images revealed that the pristine copper-based powders exhibited a spherical-like structure (Fig. 2a). After liquid-phase exfoliation, a translucent, flake-like structure with lateral dimensions ranging from 200 to 300 nm was observed by transmission electron microscopy (TEM) for copper-based metallic nanosheets (Fig. 2b). High-resolution TEM (HR-TEM) images and the corresponding fast Fourier transform (FFT) patterns (Fig. 2c) of copper-based NSs revealed that Cu nanosheets had a crystal plane spacing of 0.21 nm, assigned to the (111) plane; the CuZn nanosheets exhibited a plane spacing of 0.20 nm, corresponding to the (104) plane; the CuSn nanosheets had a plane spacing of 0.20 nm, corresponding to the (111) plane; the CuNi nanosheets had a plane spacing of 0.17 nm, corresponding to the (200) plane; and the AlCuZn nanosheets had a plane spacing of 0.20 nm, corresponding to the (310) plane. Selected-area electron diffraction (SAED) patterns indicated that the Cu, CuSn and CuNi metallenes had the original face-centered cubic structure (fcc), while the CuZn and AlCuZn metallenes were body-centered cubic (bcc) structures (Fig. 2c). X-Ray diffraction (XRD) patterns of the pristine powders and nanosheets were in accordance with the standard powder diffraction file (PDF) cards of each material (Fig. S1, ESI†). The corresponding energy dispersive spectrometer (EDS) mapping of each nanosheet confirmed the uniform elemental distribution (Fig. 2d). The atomic ratios of each nanosheet were in accordance with the standard PDF card, indicating that the liquid-phase exfoliation method did not result in the loss of raw material elements. Atomic force microscopy (AFM) revealed that the thicknesses of Cu, CuZn, CuSn, CuNi and AlCuZn were almost below 5 nm, indicating the successful preparation of five copper-based metallenes (Fig. 2e). To enhance the biocompatibility and dispersion, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethyleneglycol-2000 (DSPE-PEG2000) was used to modify the surface of copper-based metallenes. Ultraviolet-visible-near infrared (UV-vis-NIR) spectra revealed that DSPE-PEG-modified metallene suspensions exhibited strong optical absorbance (Fig. S2, ESI†), and the Tyndall effects suggested the high stability of metallene suspensions in water (Fig. S3, ESI†).

Fig. 2 Characterization of copper-based metallenes. (a) SEM images of pristine copper-based powders. (b) TEM images of copper-based metallenes. (c) HRTEM images of copper-based metallenes (inset: the corresponding FFT patterns and SAED patterns). (d) Energy dispersive spectrometer analysis of copper-based metallenes (e) AFM images and the corresponding height profiles of copper-based metallenes (units: nm).

Fenton-like activity of copper-based metallenes

Copper is a widely known element for chemodynamic therapy (CDT) due to the Fenton-like coupled electron transfer chemical reaction.^35,36 To verify the chemodynamic effect, tetramethylbenzidine (TMB) and methylene blue (MB) were used as two indicators to evaluate the Fenton-like reaction activity of copper-based metallenes. TMB changes from colorless to yellow after reaction with active ˙OH in the solution and shows distinct absorption peaks at 370 and 650 nm, which can be used as an evaluation criterion for ˙OH production (Fig. 3a). As shown in Fig. 3b, the CuZn metallene exhibited the highest ˙OH production, followed by CuSn, CuNi and Cu metallenes. Next, MB was also used to detect the ˙OH production. MB is reductive and shows a characteristic absorption peak at 664 nm (Fig. 3c). As shown in Fig. 3d, the ˙OH production capacity of the copper-based nanosheets was verified by incubating them with different concentrations of H₂O₂ (0.5, 1, 2, 4 and 8 mM). When the concentration of H₂O₂ increased, the characteristic absorption peak of MB decreased gradually, indicating that the high concentration of H₂O₂ contributed to the production of more ˙OH. Among the metallenes, the absorbances of CuZn and CuSn decreased more significantly than those of Cu and CuNi metallenes, and the absorbance of CuZn NSs decreased slightly compared to that of the CuSn metallene. In order to verify the differences in chemodynamic effects between copper-based metallenes, the steady-state kinetics of POD like enzymes were further studied. By changing the concentration of H₂O₂ and fitting the Michaelis–Menten curve, the catalytic constants K_m and V_max were obtained. The results indicate that at the same concentration, CuZn NSs have a faster enzymatic reaction rate compared to the other three copper-based metallenes (Fig. S4, ESI†). Overall, among the four Cu-based metallenes, CuZn and CuSn metallenes showed significantly higher chemodynamic effects in vitro than Cu and CuNi metallenes, and the effects of the CuZn metallene were slightly better than those of the CuSn metallene.

Fig. 3 In vitro chemodynamic properties of copper-based metallenes. (a) Colorimetric reaction of TMB with H₂O₂. (b) Absorption spectra of TMB solution containing H₂O₂ at different concentrations after mixing with copper-based metallenes. (c) MB discoloration reaction with ˙OH. (d) Absorption spectra of MB solution containing H₂O₂ at different concentrations after mixing with copper-based metallenes. (e) Colorimetric reaction of GSH with DTNB. (f) GSHOx activity (GSH consumption of copper-based NSs at different concentrations) of copper-based metallenes. (g) GSH oxidase activity (time-dependent GSH consumption of copper-based NSs in the presence of DTNB) of copper-based metallenes. (h) GSH-dependent degradation behavior of copper-based NSs. (i) Schematic diagram showing the Fenton-like reaction of copper ions. (j) 2p_1/2 and 2p_3/2 orbitals for each Cu valence state curve of Cu-based metallenes. (k) Cu ion release from copper-based NSs under different conditions.

The effectiveness of CDT is related to the amount of GSH in the microenvironment, and the Cu⁺-based Fenton-like reaction can utilize GSH in the environment to improve CDT effectiveness.³⁷ 5,5-Dithiobis-(dinitrobenzoic acid) (DTNB) was used as a GSH indicator to explore the glutathione oxidase (GSHOx) activity of copper-based metallenes (Fig. 3e). The characteristic peaks at 412 nm gradually decreased with the increase of the concentrations of the four Cu-based metallenes, among which CuZn and CuSn metallenes had a stronger GSH consumption ability compared with Cu and CuNi metallenes, and there was almost no GSH residue when the concentration of the CuZn metallene reached 50 μg mL⁻¹ (Fig. 3f). To investigate the relationship between GSH consumption and time for each copper-based metallene, each metallene was incubated with GSH, and the GSH residue was characterized at different time points using the DTNB indicator (Fig. 3g). The absorbance intensity at 412 nm gradually decreased with time, indicating an increase in GSH consumption. During 120 min incubation time, CuZn and CuSn metallenes exhibited a strong GSH depletion capacity. Among them, CuZn almost completely consumed GSH after 100 min of incubation, faster than other metallenes at the same concentration. In addition, the extent of copper ion release could potentially affect its chemodynamic effect. To simulate the degradation of the metallenes in the biofilm microenvironment, we incubated each metallene with 2 mM GSH for 2 h at a temperature of 37 °C and a pH value of 6.5. TEM images show that the CuZn metallene exhibits fragmentation and degradation, while the other metallenes do not exhibit significant effects (Fig. 3h).

In vitro chemodynamic experiments demonstrated that copper-based metallenes release copper ions to participate in the reaction. Cu²⁺ can be reduced to Cu⁺ by depleting environmental GSH, and Cu⁺ with peroxidase (POD)-like activity can continue to react with intracellular H₂O₂ to produce highly toxic ˙OH (Fig. 3i). To understand the possible mechanism and the difference in chemodynamic effects between copper-based metallenes, X-ray photoelectron spectroscopy (XPS) analysis of these four metallenes was conducted. The XPS spectra of all four metallenes revealed the presence of mixed-valence copper (Cu⁰, Cu⁺, and Cu²⁺) (Fig. 3j and Fig. S5, ESI†). The released Cu⁺ ions had the ability to catalyze the generation of hydroxyl radicals from hydrogen peroxide. The released high-valent Cu²⁺ ions from the copper-based metallene surface were expected to act as glutathione oxidase (GSHOx) to deplete intracellular GSH and be converted to Cu⁺ to enhance the chemodynamic effect. Then, the arsenazo III-coupled copper ion method was used to detect copper ion release over a specific period. The CuZn metallene released higher amounts of copper ions than the other metallenes within 2 h, both in deionized water and in physiological saline systems. In addition, the CuSn metallene exhibited a slightly higher copper ion release than CuNi and Cu metallenes (Fig. 3k).

Antibacterial performance of copper-based metallenes

Copper is one of the oldest antibacterial agents. To evaluate the antibacterial effect of copper-based metallenes, two kinds of common bacteria, Gram-positive bacteria (MRSA) and Gram-negative bacteria (Escherichia coli (E. coli)), were used. We comprehensively validated the antibacterial capacity of different groups, including antibiotics, raw metal powders, copper-based metallenes, and copper-based metallenes plus H₂O₂ groups. As shown in Fig. 4a and b, all four copper-based metallenes (Cu, CuNi, CuSn, and CuZn) showed strong antibacterial activity at low doses, and the number of MRSA colonies decreased with the increase of nanosheet concentrations (0, 0.25, 0.5, 1, and 2 μg mL⁻¹). Specifically, the CuZn metallene effectively eradicated MRSA at a concentration as low as 0.25 μg mL⁻¹, while higher concentration (1 μg mL⁻¹) was required to eradicate MRSA for the other three metallenes, indicating that the CuZn metallene showed the best antibacterial ability. The minimal inhibitory concentration (MIC) experiment was consistent with the trend of the bacterial coating experiment, and CuZn and CuSn NSs had good inhibitory effects on MRSA (Fig. S6, ESI†). Nevertheless, the antibacterial capability of all copper-based metallenes against MRSA was much higher than those of three conventional antibiotics, including levofloxacin (LVX), penicillin G (PG), and cefoxitin (FOX) (Fig. 4a and Fig. S7, ESI†). For example, almost no inhibition effect on the growth of MRSA was observed for PG and FOX groups at a concentration of 2 μg mL⁻¹. In addition, the antibacterial ability of raw metal powders was evaluated. As shown in Fig. S8 (ESI†), raw metal powders except CuZn powders exhibited a very poor antibacterial effect at a concentration of 1 μg mL⁻¹, much worse than the corresponding metallenes, indicating that the 2D engineering significantly improves the antibacterial performance of raw metal powders. Moreover, copper-based metallenes with POD-like activity can catalyze the decomposition of low amounts of H₂O₂ to produce the toxic and strong oxidant ˙OH, leading to the destruction of biological macromolecules, such as proteins and nucleic acids. Thus, the antibacterial performance of metallenes in the presence of H₂O₂ (0.1 mM) was evaluated. As depicted in Fig. 4b, the antibacterial ability of all copper-based metallenes had increased to varying degrees. For example, the CuSn NSs + H₂O₂ group had eliminated MRSA at a concentration of 0.5 μg mL⁻¹, better than the CuSn NS group (1 μg mL⁻¹), suggesting that the Cu⁺-mediated chemodynamic reaction could further enhance the antibacterial capability of metallenes.

Fig. 4 Photographs and viability of MRSA treated with copper-based metallenes in vitro. (a) Photographs of bacterial colonies of MRSA after different treatments. (b) The corresponding relative bacterial viability of MRSA (n = 3). (c) Fluorescence staining images of MRSA using SYTO 9/PI probes after different treatments (+ means plus H₂O₂, concentration units: μg mL⁻¹). One-way ANOVA was used to analyze multiple groups (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001).

Then, propidium iodide (PI, red fluorescence) and SYTO 9 (green fluorescence) were used to stain bacteria after various treatments to directly observe the dead/live bacteria (Fig. 4c and Fig. S9, ESI†). As expected, most of the MRSA strains died after being treated with each metallene, and the bactericidal effect is concentration-dependent, consistent with the previous results of plate cultivation. In addition, the staining results of bacteria treated with three antibiotics revealed that PG and FOX had almost no bactericidal effect against MRSA, while LVX exhibited a low antimicrobial activity at a relatively high concentration (2 μg mL⁻¹), suggesting the ineffectiveness of antibiotics against drug-resistant bacteria. Apart from MRSA, the antibacterial efficacy of metallenes against E. coli was evaluated. At a concentration of 0.5 μg mL⁻¹, the CuZn metallene showed a much higher bactericidal effect against E. coli (95% death), followed by the CuSn metallene (54%), CuNi metallene (57%), and Cu metallene (37%), similar to the above results against MRSA (Fig. S10A and B, ESI†). However, copper-based metallenes exhibited a weaker antibacterial ability against E. coli compared with that against MRSA, which may be attributed to the existence of outer membrane vesicles of Gram-negative E. coli.^38,39 Other similar antibacterial results against E. coli, including dead/live staining and antibacterial properties of antibiotics, were also obtained (Fig. S10C, S11 and S12, ESI†). These results clearly demonstrated that copper-based metallenes exhibited antibacterial activity that is far superior to those of raw metal powders and conventional antibiotics, and their chemodynamic antibacterial effect can be enhanced by the addition of H₂O₂.

Antibacterial mechanism of copper-based metallenes

Next, the antibacterial mechanism of copper-based metallenes was explored. As prokaryotes, bacteria do not have a complete organelle structure like eukaryotic cells, and their bacterial cell membranes are important sites closely associated with bacterial metabolism. Therefore, detecting the changes in bacterial membranes is beneficial for understanding the antibacterial mechanism of copper-based metallenes. SEM images revealed that the bacteria began to crumple and rupture after metallene treatments (4 μg mL⁻¹). The bacteria from the CuZn + H₂O₂ group showed a clear crumpled and ruptured structure (Fig. 5a). Bio-TEM images revealed that incubation with metallenes resulted in varying degrees of damage to the cell membrane, along with cytoplasmic leakage. The effect of bacterial damage was more pronounced after the addition of H₂O₂ due to the enhanced chemodynamic effect. To be noted, the CuZn + H₂O₂ group's damage was the most prominent (Fig. 5b). HR-TEM images demonstrated that CuZn and CuSn NSs attached onto the surface of bacteria and penetrated the cell wall and membrane of bacteria, which may be attributed to the sharp edge of metallenes (Fig. S13, ESI†).^40,41 Collapse and rupture of bacterial membranes induced by copper-based metallenes may result from decreased membrane potential and increased membrane permeability. For this purpose, a DiSC3(5) bacterial membrane potential fluorescent probe was used to detect the bacterial membrane potential changes (Fig. 5c). DiSC3(5) is a lipophilic fluorescent probe that enters normal cells and undergoes self-bursting in the cellular lipid layer. However, when the cell membrane is depolarized and the membrane potential is reduced, DiSC3(5) is released from within the cell membrane, resulting in an increased fluorescence signal. After the addition of copper-based metallenes, the fluorescence intensity showed a large increase in both E. coli and MRSA, indicating that Cu-based metallenes led to a decrease in the membrane potential and the bacterial membrane was in a depolarized state. Especially, the CuZn NSs + H₂O₂ group showed the strongest fluorescence. In addition, significant nucleic acid leakage was observed after copper-based metallene treatment, indicating an increase in membrane permeability (Fig. 5d). Different degrees of acid leakage were observed in both strains after treatment with metallenes. Nucleic acid leakage increased significantly in both bacterial strains after the addition of H₂O₂ due to the enhanced chemodynamic effect. CuZn NSs significantly enhanced the nucleic acid leakage, which was significantly better than those of the other copper-based metallenes. Biofilm formation is a contributing factor to bacterial resistance, as it hinders the penetration of antibiotics and offers protection for internal bacteria. Therefore, we verified the amount of bacterial biofilm after different treatments by crystal violet staining. The results showed that both E. coli and MRSA had a certain degree of reduction in the amount of the retained biofilm after 36 h of incubation with metallenes (Fig. 5e). The lowest amount of residual biofilm was observed in the CuZn NSs + H₂O₂ group.

Fig. 5 Antibacterial mechanism of copper-based metallenes. (a) SEM images of MRSA after various treatments (scale bar: 500 nm). (b) TEM images of MRSA after various treatments (scale bar: 200 nm). (c) Fluorescence intensity of DiSC3(5) in E. coli and MRSA after various treatments (n = 3). (d) Leakage of nucleic acids from differently treated E. coli and MRSA (n = 3). (e) Biofilm inhibition of E. coli and MRSA after various treatments (n = 3). (f) GSH consumption of E. coli and MRSA after various treatments (n = 3). (g) Fluorescence images of MRSA after various treatments (μg mL⁻¹). (h) Schematic diagram of the antibacterial mechanism of copper-based metallenes. One-way ANOVA was used to analyze multiple groups (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001).

We then validated the chemodynamic antibacterial capacity of copper-based metallenes by the extent of GSH depletion and ROS production within bacteria. Copper-based metallenes can catalyze the production of large amounts of ˙OH via Fenton-like reaction at low H₂O₂ concentrations, inducing oxidative stress and disrupting bacterial cell membranes. Furthermore, metallenes can also act as GSHOx to consume GSH in bacteria to enhance the catalytic antibacterial effect. Phthalic dicarboxaldehyde (OPA) was used to detect GSH content after metallene treatment to verify its chemodynamic effect. As shown in Fig. 5f, GSH content decreased after the metallene treatment. For E. coli, a significant decrease of GSH was observed for CuSn NSs and CuZn NSs while no obvious decrease was observed for Cu NSs and CuNi NSs. For MRSA, the GSH content in bacteria decreased for all groups, and the CuZn NSs + H₂O₂ group exhibited the most decrease. We then examined changes in ROS content in bacteria after different treatments using a 2,7-dichlorofluorescein diacetate (DCFH-DA) ROS fluorescent probe (Fig. 5g and Fig. S14, ESI†). No significant green fluorescence was observed in the control group. However, the green fluorescence caused by increased ROS was progressively enhanced with increasing metallene concentrations in both E. coli and MRSA. Taking CuZn NSs as an example, the fluorescence intensity showed a concentration dependence (0, 0.25, 0.5, 1, and 2 μg mL⁻¹), and the fluorescence gradually became stronger with the increase of metallene concentration. In addition, among the four copper-based metallenes, CuZn NSs and CuSn NSs produced relatively higher ROS intensities at the identical concentrations due to stronger POD-like and GSHOx activities, and the effect was even more pronounced with the addition of H₂O₂. Hence, Cu-based metallenes could slowly release Cu ions, thereby continuously depleting GSH and generating ROS, leading to oxidative stress within bacteria and causing them to undergo nucleic acid leakage and depolarization, resulting in the crumpling and rupture of bacterial membranes. In addition, the robust sheet structure of metallenes may be implanted into a bacterial membrane through mechanical force, thus effectively eradicating bacteria (Fig. 5h). The extensive surface area and rich active sites of copper-based metallenes facilitated the contact with bacteria and subsequent release of ions to effectively eliminate bacteria. The difference in antibacterial performances between different copper-based metallenes may be associated with the different release speeds and amounts of copper ions from different metallenes.

To further explore the possible antibacterial mechanism of copper-based metallenes on the molecular level, transcriptome sequencing (RNA-seq) analysis of MRSA was performed. Three groups were categorized for transcriptome analysis: PBS, Cu NSs, and CuZn NSs, and pairwise comparisons of their differential gene profiles were conducted (Fig. 6a). As depicted in the volcanic maps (Fig. 6b), 1040 differentially expressed genes (DEGs) were observed between the CuZn NSs and PBS groups. Of these, 149 gene expressions were up-regulated and 197 gene expressions were down-regulated (p < 0.05). Then, gene ontology (GO) enrichment analysis was performed to determine the functional classification of DEGs. The DEGs were assigned to 35 GO terms through classification at level 2, of which 15 terms were enriched in biological process ontology, 10 terms were enriched in cellular component ontology, and 10 terms were enriched in dative stress (Fig. 6c and Fig. S15, ESI†). Moreover, we observed the upregulation of copper-related genes, such as copper ion transport (GO:0006825), copper ion binding (GO:0005507), and stress response to copper ions (GO:1990169). This suggests that CuZn NSs can exhibit antibacterial effects by releasing copper ions. In addition, we noted the upregulation of genes associated with oxidative phosphorylation (GO:0006119) and the electron transport chain (GO:0022900). Out of the 30 KEGG pathways within categories, such as environmental information processing, genetic information processing, human disease, metabolism, and biological systems, 10 showed significant upregulation. The ribosomal and glycolysis/gluconeogenesis pathways were particularly prominent (Fig. S16, ESI†). It is noteworthy that CuZn NSs influenced oxidative phosphorylation and the electron transport chain, as evidenced by the upregulation of the three cytochrome c oxidases: QoxD, QoxC, and QoxB (Fig. S17, ESI†). To assess the variance in antimicrobial efficacy between CuZn NSs and other copper-based metallenes, we compared the distinctions between Cu NSs and CuZn NSs. First, we found that genes related to sugar metabolism were significantly downregulated. This indicates that CuZn NSs had a more significant effect on normal metabolism of bacteria than Cu NSs. In addition, significant upregulation of N-acetyltransferase activity and N-acyltransferase activity of Cu NSs may indicate the effect of CuZn NSs on bacterial drug resistance (Fig. 6d).⁴² These results suggest that the antibacterial properties of copper-based metallenes at the molecular level are achieved by the release of copper ions to induce oxidative stress within bacteria and are able to reduce bacterial drug resistance to some extent.

Fig. 6 The RNA-seq analysis of MRSA after different treatments. (a) Heat map plots for DEGs (n = 3). (b) Volcano plots of DEG (differentially expressed gene) distribution. (c) Gene ontology (GO) classification of DEGs. Oxidoreductase activity: (I) acting on a sulfur group of donors, NAD(P) as the acceptor; (II) acting on superoxide radicals as the acceptor; (III) acting on diphenols and related substances as donors; (IV) acting on diphenols and related substances as donors and oxygen as the acceptor. (d) Rich distribution point diagram of DEGs.

In vivo antibacterial and wound healing effects of copper-based metallenes

Before in vivo evaluation, the toxicity of metallenes was evaluated by using HUVEC cells. The cell viabilities of all types of copper-based metallenes were above 80% at low concentrations (0, 0.25, 0.5, 1, and 2 μg mL⁻¹) (Fig. S18, ESI†), indicating the low cytotoxicity of copper-based metallenes at low concentrations. Inspired by the excellent antibacterial effect of copper-based metallenes in vitro, we further evaluated their performance in a drug-resistant bacteria-infected wound healing model. Nine groups (PBS, LVX, FOX, PG, Cu NSs, Cu NSs + H₂O₂, CuSn NSs + H₂O₂, CuZn NSs, and CuZn NSs + H₂O₂) were established to validate the antibacterial properties of copper-based metallenes and clinical antibiotics (Fig. 7a). MRSA was injected into the back wounds of mice to induce the infection model, followed by different treatments at a fixed time. Compared to the PBS group, each copper-based metallene treatment group showed a gradual decrease in the wound area and a gradual disappearance of crusts and scars (Fig. 7b and c). On the 8th day, the wounds of all the metallene treatment groups were almost completely healed. CuZn NSs + H₂O₂ exhibited the best treatment efficiency and achieved a wound healing of ∼95%, followed by ∼90% in the CuSn NSs + H₂O₂ group (Fig. 7d). To further assess the wound antimicrobial capacity of metallenes, wound tissues were collected on the first day after treatment and the surviving MRSA was cultured on agar plates. The results indicated that the three copper-based metallenes (Cu, CuSn, and CuZn) all had a certain bactericidal effect against wound bacteria, while the bactericidal effect of antibiotics was limited (Fig. 7e and f). The bactericidal efficacy of CuZn NSs was superior to those of Cu and CuSn NSs, with the ability to eliminate more than 90% of bacteria. After adding H₂O₂, the CuZn NSs + H₂O₂ and CuSn NSs + H₂O₂ treatment groups almost eliminated MRSA, indicating that the chemodynamic antibacterial ability of metallenes was enhanced after H₂O₂ addition. In addition, pathological sections from different groups were obtained for hematoxylin–eosin (H&E) and Masson staining (Fig. 7g). Due to the poor antibacterial effect and lack of wound healing capacity, no significant healing was observed, and an incomplete epidermal layer was present in the control group. Among the three antibiotic treatment groups (LVX, FOX, and PG), only the LVX group showed a certain degree of wound tissue recovery. All other copper-based metallene treatment groups exhibited varying degrees of healing. Among them, CuZn NSs + H₂O₂ showed the best wound healing. These results were in accordance with the above antibacterial performance of metallenes in vitro. In addition, the performance of H₂O₂ in the wound healing model of drug-resistant bacterial infections was also validated. There was no significant difference in the degree of wound healing between H₂O₂ and PBS treatments (Fig. S19a–c, ESI†). H₂O₂ cannot effectively inhibit MRSA on the surface of the wound (Fig. S19d, ESI†). In addition, pathological sections from different groups were obtained for H&E and Masson staining (Fig. S19e, ESI†). Due to the poor antibacterial effect and insufficient wound healing ability, there was no significant difference between the H₂O₂ group and the PBS group, and the epidermal layer was incomplete.

Fig. 7 Antibacterial activity in vivo. (a) Schematic illustration of the MRSA-infected wound healing process. (b) Therapeutic effects on MRSA-infected BALB/c mice after different treatments. (c) Wound size radar images at different times. (d) Relative wound size (n = 3). (e) MRSA on LB agar grew from the infected wound tissue at the end of different treatments. (f) Relative bacterial survival rate of different treatments (n = 3). (g) H&E and Masson staining of the infected tissues. One-way ANOVA was used to analyze multiple groups (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001).

The body weight of all mice increased steadily within 8 days, indicating that metallenes did not affect the normal growth of the mice (Fig. S20, ESI†). To further verify the potential toxicity of copper-based metalloids in blood and organs in vivo, we assessed the presence of inflammation in wounds of mice by measuring the levels of inflammatory factors in both the wound tissue and blood samples. The results showed that the levels of TNF-α and IL-6 in the tissues and blood of the treatment groups were not significantly different from those of the control group. This indicates that the copper-based materials did not have a significant effect on inflammation in mice (Fig. S21, ESI†). We tested the blood parameters (Fig. S22, ESI†) as well as H–E staining of each major organ (heart, liver, spleen, lungs, and kidneys) (Fig. S23, ESI†). These results did not reveal any significant histological abnormalities or adverse effects, indicating that copper-based metallenes had good biosafety in vivo. In addition, long-term animal safety experiments were conducted on copper based metallenes. Subcutaneous injection of copper-based metallenes had no effect on the skin of normal mice, nor did it have significant effects on major organs, whether at 15 or 30 days (Fig. S24, ESI†). The above results collectively validate that copper-based metallenes could serve as safe and powerful antibacterial nanoagents for accelerating wound healing.

Conclusions

In conclusion, a small library of 2D copper-based metallenes with mixed-valence copper were prepared by liquid-phase exfoliation. By means of dimensional and alloy engineering, copper-based metallenes exhibited superior antibacterial capability and could effectively eliminate bacteria even at 1 μg mL⁻¹ through chemodynamic therapy and oxidative stress. When used for the treatment of MRSA-infected wound mice, the CuZn metallene quickly promoted wound healing, much better than three kinds of conventional antibiotics. This work expands the category of emerging two-dimensional metallenes and provides insights into the design of copper-based antibacterial nanoagents.

Experimental

Materials

99.9% copper powder was obtained from Aladdin Industrial Co. Ltd, 99.9% bronze powder and brass powder were purchased from Alfa Aesar, Devarda's alloy powder was purchased from Aladdin Industrial Co. Ltd, CuNi alloy powder was purchased from Chunxun Factory Area1#, Guangbo Group. 3,3′-Dipropylthiadicarbocyanine iodide [diSC3(5)] was obtained from Aladdin Industrial Co. Ltd. DSPE-PEG2000 was purchased from Shanghai Yarebio Co., Ltd. 5,5′-Dithio bis-(2-nitrobenzoic acid) (DTNB) was purchased from Aladdin Industrial Co. Ltd. Phthalaldehyde (OPA) was purchased from Beijing Solarbio Science & Technology Co., Ltd. LB-agar and LB-broth were obtained from Mab-Venture Biopharm Co., Ltd. The LIVE/DEAD^TM BacLight^TM Bacterial Viability Kit was purchased from Thermo Fisher Scientific Inc. DCFH-DA was purchased from the Beyotime Biotechnology Company. Mouse TNF-α and IL-6 ELISA kits were purchased from Wuhan Jiyinmei Biotechnology Co., Ltd.

Synthesis of copper-based metallenes

A combination of probe sonication and water bath sonication was used to prepare copper-based metallenes using isopropyl alcohol (IPA) as the solvent.^43–45 First, pristine copper-based powders (500 mg) were soaked into liquid nitrogen for 1 h to tune their mechanical properties at ultralow temperature.⁴⁶ Afterwards, the pretreated powder was dispersed into 30 mL of IPA solution, followed by probe sonication (600 W, 50%, time 3 s, interval 3 s) for 0.5 h and water bath sonication for 0.5 h (150 W), respectively. The ultrasound process was repeated 12 times. After sonication treatment, the above metallene-containing dispersions were centrifuged (4000 rpm, 20 min) 2 times to remove the unexfoliated particles and the larger and thick nanosheet components. Finally, copper-based metallenes were obtained by centrifugation of the supernatant (15 000 rpm, 15 min).

For the preparation of DSPE-PEG-modified metallenes, 50 mg of DSPE-PEG was dispersed in 2 mL of ethanol and then mixed with 2 mL of freshly prepared metallene dispersions (400 μg mL⁻¹), followed by ultrasonication under a water bath for 30 min. The above dispersion was centrifuged at 15 000 rpm for 10 min and washed with deionized water three times to remove the excess DSPE-PEG. The final DSPE-PEG-modified metallenes were stored in deionized water at 4 °C for further experiments.

Characterization

The morphology of copper-based raw powder was investigated by SEM. The morphology and thickness of the obtained copper-based metallenes were investigated by TEM and AFM. Samples were prepared by drying a drop of the relevant dispersion on a carbon-coated copper grid or mica sheet at room temperature. Crystal structures and SAED patterns of copper-based metallenes were investigated by TEM. UV-vis absorption spectra were recorded using a UV-vis spectrophotometer (Hitachi U-5100). The elemental valencies and crystal structures of the obtained copper-based metallenes were determined by XPS (ESCALAB250Xi) and XRD (X-Pert PRO MPD).

Copper ion release test

The copper-based metallenes (50 μg mL⁻¹) were dispersed in deionized water or saline and allowed to stand at 37 °C for 0, 40, 80 and 120 min. After that, the copper ion solution (10 μL) was mixed with arsenazo III buffer (pH 2.8), and the absorbance value at 630 nm was measured.

Glutathione depletion assay of copper metallenes

A UV spectrophotometric method based on 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB) was used to measure the glutathione (GSH) depletion capacity of copper-based nanosheets.⁴⁷ The procedure is as follows: 1 mM GSH was mixed with copper-based nanosheets with varying concentrations in PBS solution (pH 8.0). The mixture was then stirred at 37 °C for 2 h and subsequently centrifuged to obtain a 100 μL sample. The sample was then transferred to 900 μL of PBS solution (pH 8.0) and 4 μL of DTNB (10 mg mL⁻¹) was added to deplete –SH in GSH. The solution's absorbance change was measured at 412 nm. To measure the consumption of GSH by copper-based nanosheets at various intervals, 50 μg mL⁻¹ nanosheets were added to GSH and the reaction solutions were extracted at 0, 20, 40, 60, 80, 100, and 120 min.

In vitro assessment of chemodynamic properties of copper-based metallenes

The Fenton-like properties of copper-based nanosheets were first evaluated by their ability to degrade methylene blue (MB) in the presence of H₂O₂.⁴⁸ Copper-based nanosheets (50 μg mL⁻¹), H₂O₂ (0, 0.5, 1, 2, 4 and 8 mM), and MB (10 μg mL⁻¹) were added into PBS (pH 7.4). The mixture was then incubated under darkness for 0.5 h, followed by centrifugation (14 000 rpm, 5 min) to determine the supernatant's absorbance at 664 nm. In addition, the ability of copper-based nanosheets to produce ROS was tested using TMB.⁴⁹ TMB (0.5 mM) and copper-based nanosheets (50 μg mL⁻¹) with varying concentrations of H₂O₂ were added into PBS (pH 4.0) at room temperature. The mixture was then incubated under darkness for 0.5 h, followed by centrifugation (14 000 rpm, 5 min) to determine the supernatant's absorbance at 370 and 650 nm.

In vitro antibacterial assays

When the bacteria pre-cultured in Luria–Bertani (LB) broth at 37 °C were in the logarithmic growth phase, a mixture of 180 μL of copper-based metallenes and 20 μL of bacterial suspensions (1 × 10⁷ CFU mL⁻¹) was added to each hole of the 96-well plate. In the standard plate counting assay, 100 μL of each sample well was spread on an LB-agar. After incubation at 37 °C for 18–36 h, the colonies formed on the surface were counted to evaluate the concentration of bacteria. In live/dead staining assays, the bacteria after various treatments were stained by SYTO 9/PI. 1 × 10⁶ CFU mL⁻¹ of MRSA were incubated with LB broth containing different concentrations of copper-based metallenes (0.03125, 0.0625, 0.125, 0.25, 0.5, 1, 2, and 4 μg mL⁻¹) for 24 hours. Then, the resazurin indicator was added to each well and incubated for 4 hours. The MICs of copper-based metallenes were determined by the color change.

Biofilm assay

Firstly, 200 μL of MRSA or E. coli (10⁷ CFU mL⁻¹) in the 96-well plates were incubated at 37 °C for 24 h to form biofilm, followed by the separation of the bacterial suspension from each well. Then, PBS buffer and copper-based metallenes were added into different wells. After incubation for another 4 h, the biofilm was fixed with methanol for 30 min, followed by the addition of 0.1% crystal violet into each well and incubation in a dark environment to stain the biofilm. Finally, after dissolving the dye in 95% ethanol, the biofilm biomass was determined by measuring the absorbance of each well at 590 nm.⁵⁰

Verification of bacterial membrane damage

The change in the membrane potential of bacteria was measured using 3,3′-dipropylthiadicarbocyanine iodide [DiSC3(5)]. In detail, 4 μM DiSC3(5) and 100 mM KCl were added into 10⁶ CFU mL⁻¹ bacterial suspension, followed by incubation for 1 h at 37 °C. Then, PBS buffer or copper-based metallenes were added to the above suspension for 4 h-incubation. The fluorescence intensity of emission wavelengths of 670 nm was measured at an excitation wavelength of 620 nm.

Nucleic acid leak detection

The bacteria were cultured to the logarithmic growth phase. The sample was washed three times with sterile saline by centrifugation (6000 rpm, 5 min) and concentrated in saline to 2 × 10¹¹ CFU mL⁻¹. The sample was mixed with the bacterial solution in a 1.5 mL centrifuge tube (180 μL + 20 μL) and incubated at 37 °C for 4 h. After incubation, the supernatant was removed by centrifugation (6000 rpm, 5 min). The integrity of the bacterial cell membrane was examined by UV-vis spectroscopy at 260 nm.⁵¹

GSH consumption

The bacteria were cultured to the logarithmic growth phase. The bacterial solution was washed three times by centrifugation with sterile saline (6000 rpm, 5 min) and concentrated to 2 × 10¹¹ CFU mL⁻¹ with saline. The sample was mixed with the bacterial solution in a 1.5 mL centrifuge tube (180 μL + 20 μL), followed by incubation for 3–5 minutes at 37 °C for 4 h. After incubation, 100 μL of prepared perchloric acid lysis solution (lysis on ice for 15 min, 1%) was added to each tube. After lysis, 100 μL of the sample was removed from each tube and added to 900 μL of Tris–HCl buffer containing OPA (Tris-HCl 800 μL + OPA 100 μL, 1 mg mL⁻¹) for 90 min (120 rpm, 37 °C) on a shaker in a dark environment. After incubation, the supernatant was centrifuged (12 000 rpm, 10 min) and tested using a fluorescence spectrophotometer (excitation wavelength 350 nm, emission wavelength 420 nm, excitation voltage 400 V, slit width 10 nm).⁵²

Detection of intracellular ROS

2′,7′-Dichlorofluorescin diacetate (DCFH-DA, Beyotime Biotechnology Company) was utilized to detect the ROS in bacteria. In brief, bacteria (10⁸ CFU mL⁻¹) after metallene treatments were incubated with 20 × 10⁻⁶ M DCFH-DA at 37 °C for 30 min in the dark and then observed using a fluorescence microscope.

In vitro biocompatibility assessment of copper-based metallenes

HUVECs were seeded onto plates with a density of 1 × 10⁴ cells per well and incubated for 24 h. After removing the medium, 200 μL of Cu NSs, CuZn NSs, CuSn NSs, CuNi NSs, PG, LVX, and FOX (concentrations ranging from 0 to 2 μg mL⁻¹) dispersed in fresh DMEM were added to each well and incubated for another 24 h. To ensure consistency, three parallel wells were set up for each concentration. Afterwards, the culture medium was removed and the cells were rinsed twice with new DMEM medium. For the MTT assay, 20 μL of MTT solution (5 mg mL⁻¹ in sterile PBS) and 150 μL of fresh DMEM medium were added. After 4 h of incubation, 150 μL of a strong solvent, DMSO, was added. The formazan was dissolved by incubation for 15 minutes under dark conditions after gentle shaking. Finally, the absorption at 490 nm was measured.

RNA-seq assay

1 mL of bacterial (MRSA) suspension at the log phase (OD 600 = 0.5–0.8) was placed in a 1.5 mL enzyme-free tube. After centrifugation at 4 °C, 10 000 rpm for 2 min, the supernatant was completely removed. The centrifugation process was repeated multiple times to collect the bacterial cells in several tubes. The bacteria were quickly frozen in liquid nitrogen for 15 minutes and stored at −80 °C. Finally, the transcriptome sequencing was performed at Shanghai Personalbio technology Co. Ltd.

Wound healing in vivo

Typically, six-week-old female BALB/c mice were randomly divided into PBS, LVX, PG, FOX, Cu NSs, Cu NSs + H₂O₂, CuZn NSs, CuZn NSs + H₂O₂, CuSn NSs and CuSn NSs + H₂O₂ groups (n = 8 for each group). The back hair of each mouse was shaved off and a wound with a diameter of ∼1 cm was formed by a surgical procedure. The skin wounds were preprocessed with MRSA suspensions (1 × 10¹¹ CFU mL⁻¹) for 24 h. After one day, the infected wound area was uniformly sprayed with 200 μL of the solutions PBS, LVX, PG, FOX, Cu NSs, Cu NSs + H₂O₂, CuZn NSs, CuZn NSs + H₂O₂, CuSn NSs or CuSn NSs + H₂O₂. Subsequently, the wounds were photographed on days 2, 4, 6, and 8. Changes in wound sizes were measured. Three mice per group were euthanized on days 0 and 8. For histological analysis, immunohistochemical staining of hematoxylin and eosin (H&E) and Masson's trichrome staining (Masson) were performed; the skin of the wounds was excised and fixed with a tissue fixative. On day 8, blood samples were obtained from the fundus artery of the mice and centrifuged at 3000 rpm for 5 min. The collected serum was then used for blood biochemical and blood routine tests.

All animal procedures were performed in accordance with ARRIVE guidelines and experiments were approved by the Institutional Animal Care and Use Committee of Hefei University of Technology (No. HFUT20221121001).

Statistical analysis

The obtained data were expressed as the mean value ± standard deviation, and the statistical significance between the two groups was analyzed by the one-way ANOVA method. *p < 0.05 was considered statistically significant, **p < 0.01 was considered significant, and ***p < 0.001 was considered extremely significant.

Data availability

The data supporting this article have been included within the main text and ESI.†

Conflicts of interest

There are no conflicts of interest to declare.

Acknowledgements

This work was financially supported by the National Natural Science Foundation of China (No. 82270584, 52073313 and 52272275), the University Synergy Innovation Program of Anhui Province (No. GXXT-2022-060), and the Fundamental Research Funds for the Central Universities (JZ2024HGTG0320).

Notes and references

1. O. Bubnova, Nat. Nanotechnol., 2018, 13, 272.
2. P. Prabhu and J.-M. Lee, Chem. Soc. Rev., 2021, 50, 6700–6719.
3. Z. Wei, Z. Zhao, C. Qiu, S. Huang, Z. Yao, M. Wang, Y. Chen, Y. Lin, X. Zhong, X. Li and J. Wang, Nat. Commun., 2023, 14, 661.
4. C. Lu, R. Li, Z. Miao, F. Wang and Z. Zha, Chem. Soc. Rev., 2023, 52, 2833–2865.
5. M. Xie, S. Tang, B. Zhang and G. Yu, Mater. Horiz., 2023, 10, 407–431.
6. J. Fan, Z. Feng, Y. Mu, X. Ge, D. Wang, L. Zhang, X. Zhao, W. Zhang, D. J. Singh, J. Ma, L. Zheng, W. Zheng and X. Cui, J. Am. Chem. Soc., 2023, 145, 5710–5717.
7. W. Peng, J. Zhou, Y.-R. Lu, M. Peng, D. Yuan, T.-S. Chan and Y. Tan, Proc. Natl. Acad. Sci. U. S. A., 2023, 120, e2222096120.
8. N. Briggs, B. Bersch, Y. Wang, J. Jiang, R. J. Koch, N. Nayir, K. Wang, M. Kolmer, W. Ko, A. De La Fuente Duran, S. Subramanian, C. Dong, J. Shallenberger, M. Fu, Q. Zou, Y.-W. Chuang, Z. Gai, A.-P. Li, A. Bostwick, C. Jozwiak, C.-Z. Chang, E. Rotenberg, J. Zhu, A. C. T. van Duin, V. Crespi and J. A. Robinson, Nat. Mater., 2020, 19, 637–643.
9. M. A. Steves, Y. Wang, N. Briggs, T. Zhao, H. El-Sherif, B. M. Bersch, S. Subramanian, C. Dong, T. Bowen, A. D. L. Fuente Duran, K. Nisi, M. Lassaunière, U. Wurstbauer, N. D. Bassim, J. Fonseca, J. T. Robinson, V. H. Crespi, J. Robinson and K. L. Knappenberger Jr, Nano Lett., 2020, 20, 8312–8318.
10. S. Rajabpour, A. Vera, W. He, B. N. Katz, R. J. Koch, M. Lassaunière, X. Chen, C. Li, K. Nisi, H. El-Sherif, M. T. Wetherington, C. Dong, A. Bostwick, C. Jozwiak, A. C. T. van Duin, N. Bassim, J. Zhu, G.-C. Wang, U. Wurstbauer, E. Rotenberg, V. Crespi, S. Y. Quek and J. A. Robinson, Adv. Mater., 2021, 33, 2104265.
11. J. Zhang, F. Lv, Z. Li, G. Jiang, M. Tan, M. Yuan, Q. Zhang, Y. Cao, H. Zheng, L. Zhang, C. Tang, W. Fu, C. Liu, K. Liu, L. Gu, J. Jiang, G. Zhang and S. Guo, Adv. Mater., 2022, 34, 2105276.
12. Y. Liu, Y. Xiao, Y. Cao, Z. Guo, F. Li and L. Wang, Adv. Funct. Mater., 2020, 30, 2003196.
13. N. Tao, Z. Zeng, Y. Deng, L. Chen, J. Li, L. Deng and Y.-N. Liu, Chem. Eng. J., 2023, 456, 141109.
14. C. Feng, J. Ouyang, Z. Tang, N. Kong, Y. Liu, L. Fu, X. Ji, T. Xie, O. C. Farokhzad and W. Tao, Matter, 2020, 3, 127–144.
15. B. Jiang, Y. Guo, F. Sun, S. Wang, Y. Kang, X. Xu, J. Zhao, J. You, M. Eguchi, Y. Yamauchi and H. Li, ACS Nano, 2023, 17, 13017–13043.
16. Y. Liu, K. N. Dinh, Z. Dai and Q. Yan, ACS Mater. Lett., 2020, 2, 1148–1172.
17. V. Kochat, A. Samanta, Y. Zhang, S. Bhowmick, P. Manimunda, S. A. S. Asif, A. S. Stender, R. Vajtai, A. K. Singh, C. S. Tiwary and P. M. Ajayan, Sci. Adv., 2018, 4, e1701373.
18. M. Luo, Z. Zhao, Y. Zhang, Y. Sun, Y. Xing, F. Lv, Y. Yang, X. Zhang, S. Hwang, Y. Qin, J.-Y. Ma, F. Lin, D. Su, G. Lu and S. Guo, Nature, 2019, 574, 81–85.
19. M. Xie, S. Tang, Z. Li, M. Wang, Z. Jin, P. Li, X. Zhan, H. Zhou and G. Yu, J. Am. Chem. Soc., 2023, 145, 13957–13967.
20. D. B. Miracle, Nat. Commun., 2019, 10, 1805.
21. G. Grass, C. Rensing and M. Solioz, Appl. Environ. Microbiol., 2011, 77, 1541–1547.
22. M. Vincent, P. Hartemann and M. Engels-Deutsch, Int. J. Hyg. Environ. Health, 2016, 219, 585–591.
23. C. Cao, X. Wang, N. Yang, X. Song and X. Dong, Chem. Sci., 2022, 13, 863–889.
24. M. Vincent, R. E. Duval, P. Hartemann and M. Engels-Deutsch, J. Appl. Microbiol., 2018, 124, 1032–1046.
25. I. Salah, I. P. Parkin and E. Allan, RSC Adv., 2021, 11, 18179–18186.
26. J. Qian, Q. Dong, K. Chun, D. Zhu, X. Zhang, Y. Mao, J. N. Culver, S. Tai, J. R. German, D. P. Dean, J. T. Miller, L. Wang, T. Wu, T. Li, A. H. Brozena, R. M. Briber, D. K. Milton, W. E. Bentley and L. Hu, Nat. Nanotechnol., 2023, 18, 168–176.
27. C. A. P. Bastos, N. Faria, J. Wills, P. Malmberg, N. Scheers, P. Rees and J. J. Powell, NanoImpact, 2020, 17, 100192.
28. D. J. Horton, H. Ha, L. L. Foster, H. J. Bindig and J. R. Scully, Electrochim. Acta, 2015, 169, 351–366.
29. C. L. Trybus, R. P. Vierod and P. W. Robinson, US Pat., 12/943, 196, 2010.
30. J. Lee, H. Liao, Q. Wang, J. Han, J.-H. Han, H. E. Shin, M. Ge, W. Park and F. Li, Exploration, 2022, 2, 20210086.
31. S. He, M. Lin, Q. Zheng, B. Liang, X. He, Y. Zhang, Q. Xu, H. Deng, K. Fan and W. Chen, Adv. Healthcare Mater., 2024, 13, 2303548.
32. S. Ji, B. Jiang, H. Hao, Y. Chen, J. Dong, Y. Mao, Z. Zhang, R. Gao, W. Chen, R. Zhang, Q. Liang, H. Li, S. Liu, Y. Wang, Q. Zhang, L. Gu, D. Duan, M. Liang, D. Wang, X. Yan and Y. Li, Nat. Catal., 2021, 4, 407–417.
33. C. Zhang, Y. Xu, P. Lu, C. Wei, C. Zhu, H. Yao, F. Xu and J. Shi, Angew. Chem., Int. Ed., 2019, 58, 8814–8818.
34. F. Wang, Z. Wang, T. A. Shifa, Y. Wen, F. Wang, X. Zhan, Q. Wang, K. Xu, Y. Huang, L. Yin, C. Jiang and J. He, Adv. Funct. Mater., 2017, 27, 1603254.
35. Y. Bao, H. Zhang, X. Wu, R. Yan, Z. Wang, C. Guo and Y. Jin, ACS Appl. Nano Mater., 2023, 6, 14410–14420.
36. Y.-N. Hao, Y.-R. Gao, Y. Li, T. Fei, Y. Shu and J.-H. Wan, Adv. Mater. Interfaces, 2021, 8, 2101173.
37. Z. Tang, Y. Liu, M. He and W. Bu, Angew. Chem., Int. Ed., 2019, 58, 946–956.
38. H. M. Kulkarni, R. Nagaraj and M. V. Jagannadham, Microbiol. Res., 2015, 181, 1–7.
39. N. Furuyama and M. P. Sircili, BioMed Res. Int., 2021, 2021, 1490732.
40. R. P. Pandey, P. A. Rasheed, T. Gomez, K. Rasool, J. Ponraj, K. Prenger, M. Naguib and K. A. Mahmoud, ACS Appl. Nano Mater., 2020, 3, 11372–11382.
41. K. Rasool, K. A. Mahmoud, D. J. Johnson, M. Helal, G. R. Berdiyorov and Y. Gogotsi, Sci. Rep., 2017, 7, 1598.
42. S. Ahmed, S. A. Sony, M. B. Chowdhury, M. M. Ullah, S. Paul and T. Hossain, Sci. Rep., 2020, 10, 19381.
43. T. Chen, Z. Cheng, Q. Tian, J. Wang, X. Yu and D. Ho, ACS Appl. Nano Mater., 2020, 3, 6440–6447.
44. D. Gopalakrishnan, D. Damien and M. M. Shaijumon, ACS Nano, 2014, 8, 5297–5303.
45. X. Li, W. Li, Z. Miao, C. Lu, H. Ma, Y. Xu, D. Gong, C.-Y. Xu and Z. Zha, J. Mater. Sci. Technol., 2023, 148, 186–198.
46. Y. Wang, Y. Liu, J. Zhang, J. Wu, H. Xu, X. Wen, X. Zhang, C. S. Tiwary, W. Yang, R. Vajtai, Y. Zhang, N. Chopra, I. N. Odeh, Y. Wu and P. M. Ajayan, Sci. Adv., 2017, 3, e1701500.
47. N. Xu, A. Hu, X. Pu, J. Wang, X. Liao, Z. Huang and G. Yin, J. Mater. Chem. B, 2022, 10, 3104–3118.
48. R. Cao, W. Sun, Z. Zhang, X. Li, J. Du, J. Fan and X. Peng, Chin. Chem. Lett., 2020, 31, 3127–3130.
49. X. Zhou, Y. Qi, Y. Tang, H. Gao, L. Lv, X. Lei, L. Hu and Z. Yan, Microchim. Acta, 2022, 189, 314.
50. Z. Xu, Y. Liang, S. Lin, D. Chen, B. Li, L. Li and Y. Deng, Curr. Microbiol., 2016, 73, 474–482.
51. M. Singh, A. K. Mallick, M. Banerjee and R. Kumar, Bull. Mater. Sci., 2016, 39, 1871–1878.
52. X. Kang, F. Bu, W. Feng, F. Liu, X. Yang, H. Li, Y. Yu, G. Li, H. Xiao and X. Wang, Adv. Mater., 2022, 34, 2206765.

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4mh01175a

‡ These authors contributed equally.

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