Green solid-state synthesis of Cu4O3/biochar composites with high antimicrobial activity

doi:10.21203/rs.3.rs-4879778/v1

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Green solid-state synthesis of Cu₄O₃/biochar composites with high antimicrobial activity

https://doi.org/10.21203/rs.3.rs-4879778/v1

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Infectious diseases caused by pathogenic microorganisms have inflicted significant calamities upon human society. The Cu₄O₃/biochar composites with high efficacy of antimicrobial properties are developed for the first time via a green solid synthesis strategy of ball milling and sintering processes in this work. The mechanistic investigation revealed that the biochar, e.g. corn stover, plays multiple physiochemical roles as a support carrier, dispersant, and reducing agent, regulating elegantly the stoichiometric ratio of Cu₂O and CuO. The potent antimicrobial activities of the Cu₄O₃/biochar composite were confirmed against E. coli, S. aureus, and methicillin-resistant Staphylococcus aureus MRSA through minimum inhibitory concentration (MIC) testing, exhibiting extremely low MIC values against the Gram-positive strains S. aureus and MRSA. Furthermore, the antibacterial mechanisms of the Cu₄O₃/biochar composite were studied through the experiments and quantum mechanical computations, showing the enhanced effects between the successive release of Cu(I)/Cu(II) ions and the generation of reactive oxygen species. This work reports the first solid-state symproportionation reactions of CuO_x to prepare high-purity Cu₄O₃, stabilized by biochar with the advantages of simplicity, low cost, brevity, mild reaction conditions, and eco-friendliness. The Cu₄O₃/biochar composite can be applied as additives in anti-bacterial materials to protect against harmful microbial infections including superbugs.

Materials Chemistry

Cu4O3/biochar composite

solid-state synthesis

symproportionation reaction

antimicrobial material

superbug

Infectious diseases caused by pathogenic microorganisms have brought severe mortality, social issues, and economic losses to human society.^[1] Antimicrobial resistance (AMR) induced by the use or misuse of antibiotics, has emerged as one of the foremost global threats to public health and development.^[2] In 2019, AMR caused directly 1.27 million deaths worldwide, with an additional 4.95 million deaths attributed indirectly.^[3] Failure to take sufficient action to control AMR is forecasted to escalate annual mortality to approximately 10 million by 2050.^[2b]

Copper oxides (Cu_xO), such as Cu₂O and CuO have demonstrated significant potential in antimicrobial applications against pathogenic microorganisms due to their high efficiency antimicrobial, antifungal, and antiviral properties as well as their low costs and environment benign.^[4] Cu₄O₃, also known as paramelaconite, was initially discovered in the 1870s at the Copper Queen Mine in Bisbee, Arizona, USA, and represents one of the least studied Cu_xO.^[5] Its unique structure exhibits the characteristics with a mixed copper valence states and lattice positioned between Cu₂O and CuO, constituting a metastable intermediate compound containing both Cu(I) and Cu(II) ions.^[6]

After more than 100 years since its initial discovery, the explicit synthetic pathway for Cu₄O₃ was first reported in 1996: Morgan et al. prepared Cu₄O₃ by extracting copper or its oxides with concentrated ammonia in a Soxhlet apparatus. However, the quantitative analysis revealed that the synthesized product consisted of 35% Cu₄O₃, 27% Cu₂O, and 38% CuO. The low purity of Cu₄O₃ may be attributed to the challenge of stabilizing both Cu(I) and Cu(II) ions in the solution system.^[5] Additionally, Cu₄O₃ is thermodynamically unstable and can decompose easily into Cu₂O and CuO during the preparation process.^[6–7] Subsequent studies have explored gas-phase synthesis methods, such as chemical vapor deposition (CVD) or sputtering deposition, for preparing pure phases of Cu₄O₃ or partially containing Cu₄O₃.^[8] For instance, Blobaum et al. reported the preparation of Cu₄O₃ thin films by sputter deposition on CuO targets. However, the yield was only at the milligram level, and the morphology and purity of the products were significantly affected by factors such as oxygen partial pressure and substrate temperature.^[8a] The other studies prepared Cu₄O₃ or its composites by liquid phase chemistry methods such as solvothermal or wet chemistry methods.^[9] Zhao et al. synthesized phase-pure Cu₄O₃ microspheres by the solvothermal method under solvent conditions of N, N-dimethylformamide (DMF), and ethanol with Cu(NO₃)₂ as the precursor.^[9b] However, Jiang et al. attempted to replicate Zhao's work but with less satisfactory results, yielding only impure phase Cu₄O₃ in the product.^[7] Liquid-phase chemistry for the preparation of Cu₄O₃ still faces challenges such as process complexity, lack of control, low yield, and difficulty in obtaining the high-purity Cu₄O₃. The low-cost, large-scale synthesis of high-purity Cu₄O₃ and the maintenance of their stability still pose a great challenge, hindering the further exploration of their properties and applications.

Biochar is one of the carbonaceous materials prepared through the pyrolytic carbonization of biomass, (e.g., crop and forestry residues, manure, and solid organic wastes from urban and industrial sources) in an O₂-limited/free environment.^[10] The high specific surface area, rich porous structure, oxygenated functional groups on the surface, and flaky aromatic structure confer rich physicochemical properties and application potential to biochar.^[10a] In recent years, biochar has found widespread use in soil improvement, sewage treatment, air purification, carbon sequestration, and emission reduction.^[11] Supporting various functional metal oxide particles onto biochar to synthesize biochar-based composites can combine the respective advantages of both parties, enhance the dispersion and stability, improve the application performance of the functional particles, and endow biochar with new application capabilities.^[12] In addition to the physical support and stabilization, biochar substrate exhibits a unique redox capacity from the chemistry point of view.^[13] The recent studies have focused on preparing biochar-based composites through the supporting capacity and redox property of biochar, applied to various applications such as pollutant adsorption, water treatment, and antibacterial agents.^{[12a, 14]}

In this study, we present for the first time in-situ preparation of Cu₄O₃/biochar (e.g., corn stover) composite via a solid-state synthesis strategy combining ball milling and sintering processes in air. In this preparation process, copper formate serves as the copper source, while biochar acts as the support carrier, dispersant, and reducing agent. Compared with the preparation methods of Cu₄O₃ reported previously, the solid-state preparation approach displays advantages such as facile, a concise short process flow, controllability, and mild conditions, without the need for any oxidizing or reducing chemical reagents, and little generation of solid/liquid waste, coinciding with the concept of “green chemistry” manufacture. We systematically investigated the crystal structure, purity, valence state, morphology, and loading amount of Cu₄O₃/biochar composite through various characterization methods, with the elucidation of the preparation process and mechanism. Through minimum inhibitory concentration (MIC) antibacterial testing and bacterial morphology analysis, we demonstrated the highly efficient antibacterial properties of the Cu₄O₃/biochar composite against Escherichia coli E. coli, Staphylococcus aureus S. aureus, and Methicillin-resistant Staphylococcus aureus MRSA. Notably, the Cu₄O₃/biochar composite displayed remarkably low MIC values (0.100 µg/mL and 0.125 µg/mL, respectively) against Gram-positive strains S. aureus and MRSA. Furthermore, we systematically investigated the antibacterial mechanism and the stability in solution of the Cu₄O₃/biochar composite through experimental methods coupled with calculations, demonstrating for the first time the Cu-ion precipitation mechanism of Cu₄O₃ formation. The Cu₄O₃/biochar composite is anticipated to be applied in public facilities and healthcare environments, imparting surfaces with heightened resistance to infection by pathogenic microorganisms, including superbugs. The synthesis approach developed in this work realized the first solid-state CuO_x symproportionation reactions and prepared the Cu₄O₃/biochar composites by combining ball milling and sintering processes. The solid-state symproportionation (SSS) approach can be extended to achieve large-scale preparation and facilitate more fundamental studies and potential applications of Cu₄O₃.

2.1 Preparation and characterization of the Cu₄O₃/biochar composite

Figure 1. depicts schematically the solid-state preparation process for the Cu₄O₃/biochar composite through two-stage ball millings followed by sintering processes. The corn stover biochar (CSBC) was first derived from corn stover via grinding and washing, followed by slow pyrolysis.^[15] Copper formate underwent the successive ball milling process (BM-1) and subsequent co-ball milling with the CSBC (BM-2) to obtain the copper formate/CSBC precursor. The SEM images (Figure S1, Supporting Information) illustrate that the BM-1 process refined the particle size of copper formate from the initial micron-sized flakes to uniformly distributed submicron particles.^[16] The CSBC underwent a co-ball milling process (BM-2) with copper formate resulting in the transformation of its initial irregular morphology and unevenly sized particles into small particles with uniform sizes (5–20 µm) (Figure S2a-b, Supporting Information), demonstrating the effectiveness of the ball milling process in reducing the biochar sizes.^[17] Moreover, the submicron copper formate particles served as grinding balls to assist in grinding the biochar.^[18] Furthermore, copper formate particles exhibited uniform and dense distribution on the surface of CSBC (Figure S2c-d, Supporting Information). Ultimately, the Cu₄O₃/CSBC composite was obtained via the solid-state sintering process at 230 ℃ for 12 minutes. The parameters of sintering processing were carefully optimized as discussed later.

The crystal structure of the Cu₄O₃/CSBC composite was initially analyzed using X-ray diffraction (XRD). As illustrated in Fig. 2a, CSBC exhibited typical broad and gradual amorphous carbon diffraction peaks at 2θ = 15–30°;^[19] the X-ray diffraction peaks appearing at 28.2°, 30.7°, 35.7°, 36.3°, 44.0°, 58.3°, 63.9°, 65.0°, and 75.5° can be assigned to the (112), (200), (202), (004), (220), (224), (400), (206), and (422) crystal planes of Cu₄O₃. All these diffraction peaks were consistent with the characteristic peaks of the Cu₄O₃ standard card (JCPDS No.49-1830), and no diffraction peaks from other copper compounds were observed, such as Cu, CuO, and Cu₂O, indicating that the Cu₄O₃/CSBC composite contains high purity Cu₄O₃.

X-ray photoelectron spectroscopy (XPS) was employed to further evaluate the valence states of the Cu₄O₃/CSBC composite. The presence of elemental Cu signal peaks confirmed the deposition of elemental Cu on the biochar surface, as illustrated in the XPS spectra (Fig. 2b). The Cu 2p_3/2 XPS spectrum (Fig. 2c) displays the fitted peaks at 932.8 eV and 934.8 eV attributed to Cu(I) and Cu(II) ions, respectively.^[9a] The XPS spectra confirm that Cu₄O₃ is a mixed-valence compound comprising both Cu(I) and Cu(II) ions. Moreover, the content of Cu element in the Cu₄O₃/CSBC composite was detected by the inductively coupled plasma-optical emission spectrometry (ICP-OES), the mass ratio of Cu is 12.87 wt.%, indicating the content of Cu₄O₃ is 15.30 wt.% (Table S1, Supporting Information) according to the mass ratio (84.1%) of Cu element relative to Cu₄O₃. This value coincides with our predetermined experimental value, indicating that the solid-synthesis strategy can achieve the quantitatively controllable preparation of the contents of Cu₄O₃ in the Cu₄O₃/CSBC composite.

The structural composition of the Cu₄O₃/CSBC composite was examined using Fourier-transform infrared (FTIR) spectroscopy (Fig. 2d). The Cu₄O₃/CSBC composite exhibited absorption bands resembling those of the CSBC at wavelengths of 3425 cm^-1, 2920 cm^-1, 1720 cm^-1, and 1610 cm^-1, representing -OH, C-H, -C = O, and -C = C stretching vibrations, respectively.^[20] However, the absorption bands related to -C = O and -C = C of aromatic groups appeared more pronounced, while the -CH₂ band intensity decreased compared to the characteristic absorption bands of CSBC. The escalation in oxygen-containing functional groups and the decline in methyl groups of the biochar could be attributed to intense mechanical collision during the BM-2 process and the thermal oxidation process by O₂ during the solid-state sintering process.^{[17a, 21]} Additionally, three signal bands corresponding to the Cu-O stretching vibration of Cu₄O₃ were detected at wavelengths of 616 cm^-1, 547 cm^-1, and 470 cm^-1,^[9a] further indicating the high purity of Cu₄O₃ within the Cu₄O₃/CSBC composite.

The microscopic morphology and elemental distribution of the Cu₄O₃/CSBC composite were investigated using scanning electron microscopy (SEM), transmission electron microscopy (TEM), and energy-dispersive X-ray spectroscopy (EDX) elemental mapping. The macroscopic SEM image (Fig. 2e) illustrates the uniform particle size distribution of the Cu₄O₃/CSBC composite. The microscopic SEM images (Fig. 2f-g) at various magnifications reveal the uniform and dispersed distributions of Cu₄O₃ nanoparticles that form clusters on the surface of the CSBC. The morphology of Cu₄O₃ was further elucidated by TEM analysis, confirming the cluster structures formed by many fine nanoparticles (Fig. 2h-i). The high-resolution transmission electron microscopy (HRTEM) image (Fig. 2j) displays the distinct lattice streaks with a lattice spacing of 0.25 nm corresponding to the (202) crystal plane of Cu₄O₃ crystal (JCPDS No.49-1830). The EDX elemental mapping (Fig. 2k) indicates a homogeneous spatial arrangement of Cu and O elements within the Cu₄O₃ nano-sized clusters, consistent with the TEM images.

We further investigated the influence of the preparation processing parameters, e.g. sintering temperature/time and loading capacity, on the reaction products. When the solid-state sintering was conducted at 220 ℃ for 12 min, copper formate was not completely decomposed and impurities of Cu₂O and CuO appeared in the sintered products (Figure S3a, Supporting Information). At a sintering temperature of 240 ℃, the XRD analysis reveals the presence of Cu₂O and CuO impurities in the products (Figure S3b, Supporting Information), indicating the decomposition of partial Cu₄O₃ to Cu₂O and CuO at 240 ℃, consistent with the reported transition temperature of Cu₄O₃.^[9b] Subsequently, we assessed the supporting capacity of CSBC for Cu₄O₃. When the supporting ratio of Cu₄O₃ was 10 wt.%, the solid-state sintered products exhibited pure-phase Cu₄O₃ (Figure S4a, Supporting Information); however, attempting to support 20 wt.% of Cu₄O₃ resulted in sintered products containing Cu₂O and CuO impurities (Figure S4b, Supporting Information). The appearance of impurity by-products in the sintering process may be caused by the heat released from the decomposition of copper formate that could cause the decomposition of Cu₄O₃^[22].

In order to assess the generalizability of various biochar sources for the preparation of Cu₄O₃/biochar composite, we explored the impact of biochar derived from different biomass sources (e.g., palm leaves, cedar wood, and bagasse) on the sintered products. Figure 3(a-c) displays the XRD spectra of the sintered products resulting from various biochar (named PLBC, CWBC, and BBC, respectively) prepared from palm leaves, cedar wood, and bagasse as biochar feedstock sources after the ball milling (BM-2) and sintering processes with copper formate. Among these, copper formate/PLBC precursor yielded the Cu₄O₃/PLBC composite containing pure-phase Cu₄O₃ (Fig. 3a); copper formate/CWBC precursor accommodated a support amount of 10 wt.% Cu₄O₃ without impurities (Fig. 3b_I) while attempts to support 15 wt.% Cu₄O₃ led to impurities of Cu₂O and CuO in the sintered products (Fig. 3b_II), which may be due to the differences in specific surface area and supporting capacity among the various biochar types.^[12a] At a pyrolysis temperature of 300 ℃ for bagasse biochar, the sintered product contained the impurity of Cu₂O, in addition to Cu₄O₃ (Fig. 3c_I). However, at a pyrolysis temperature of 400 ℃, the sintered product obtained showed high purity Cu₄O₃ (Fig. 3c_II). The optimal processing parameters for preparing Cu₄O₃/biochar composite from PLBC, CWBC, and BBC are listed in Table S2.

Subsequently, the morphology of the Cu₄O₃/biochar composite obtained from different sources of biochar was analyzed by SEM. As depicted in Fig. 3a₁, 3b₁, and 3c₁, the biochar inherited the morphology and characteristics of the corresponding biomass before undergoing the Cu₄O₃/biochar composite preparation process. In contrast, the morphology and structure of the sintered products were relatively similar in that Cu₄O₃ nanoparticle clusters were uniformly dispersed on the surface of the biochar particles with consistent particle sizes (Fig. 3a_2-3, 3b_2-3, and 3c_2-3). The exploration of biochar feedstock sources experiments demonstrated that the preparation process of Cu₄O₃/biochar composite through ball milling combined with sintering processes exhibits a certain degree of generalizability. Nevertheless, the functional groups, specific surface area, and supporting capacity of biochar derived from various feedstock sources,^[23] necessitating adjustments to the pyrolysis parameters or the preparation process based on the specific raw feedstock when preparing the Cu₄O₃/biochar composite. In general, all the studied Cu₄O₃/biochar composites can be prepared successfully while their optimal processing parameters may vary depending on the biochar resources.

2.2 Formation mechanism of the Cu₄O₃/CSBC composite

To investigate the formation mechanism of the Cu₄O₃/CSBC composite, we employed a rapid cooling method using liquid nitrogen to quench the chemical reactions during the process of solid-state sintering to identify intermediate phases in the evolution of sintering reactions.^[16] Fig. 4a illustrates the comparative XRD spectra of the sintered products derived from copper formate/CSBC precursor during various sintering times (0–12 min) at 230 ℃. As the sintering time prolonged, the intensity of the signal XRD peaks corresponding to copper formate diminished gradually and disappeared completely at 10 min, indicating its rapid decomposition process. Furthermore, three distinct signal peaks of Cu₄O₃, Cu₂O, and CuO emerged simultaneously during the intermediate stages. The diffraction peak intensities of Cu₂O and CuO initially increased, then weakened, and disappeared eventually at 12 min, indicating the formation and subsequent disappearance of intermediate phases involving Cu₂O and CuO. These observations discovered the formation of intermediate phases Cu₂O and CuO during the sintering process, suggesting that Cu₄O₃ should originate from Cu₂O and CuO via the solid-state symproportionation reactions between Cu₂O and CuO.^[9b] Such symproportionation reaction among Cu_xO was originally proposed in liquid phase reactions^[9b], but it is for the first time demonstrated in solid-state reactions in this work to our best knowledge.

In our previous study,^[16] we analyzed the thermal decomposition process of copper formate. Copper formate undergoes two consecutive stepwise cation reduction processes to generate Cu, which is subsequently oxidized by O₂ to form Cu₂O and CuO. However, in the absence of any carrier or medium, Cu₂O and CuO cannot directly disproportion into Cu₄O₃ in air. We further investigated the critical role of CSBC in the preparation process of Cu₄O₃/CSBC composite through a series of controlled experiments. The specifically designed processing processes are shown in Table S3 (Supporting Information), including untreated CSBC (CSBC), CSBC treated with the BM-2 process (BM-CSBC), CSBC treated with BM-2 followed by sintering processes (BM-S-CSBC), and the Cu₄O₃/CSBC composite. Figure 4b illustrates the C 1s spectra and their fitting results of CSBC after different preparation processes. Copper formate participates in the BM-2 process with CSBC, so we excluded the influence of formate groups in copper formate on the fitting results of C1s through filtration and washing after BM-2 in the controlled experiments for the samples BM-CSBC and BC-S-CSBC. As can be seen in Fig. 4b, the comparisons of C 1s among the CSBC, BM-CSBC, and BM-S-CSBC samples exhibited an increase in the contents of oxygen-containing functional groups C = O and O-C = O. The normalized quantitative analysis of C 1s spectra fitting results (Fig. 4c) further clarifies this phenomenon. Furthermore, the content of C-C/C = C/C-H functional groups of BM-CSBC, and BM-S-CSBC samples decreased compared to that of CSBC. The analysis result indicates that the strong mechanical collisions during the BM-2 process and the thermal oxidation reaction in air during the sintering process increase the oxygen-containing functional groups of biochar and decrease the abundance of C-H functional groups,^{[17a, 21]} which is consistent with the results of FTIR analysis. As a comparison, the relative contents of C = O and O-C = O in the Cu₄O₃/CSBC composite were notably higher compared with those in CSBC, BM-CSBC, and BM-S-CSBC, with a further reduction in C-H content, indicating the chemical role of biochar in additional oxidative processes, specifically participating in the redox conversion from CuO to Cu₂O.

Based on the analysis results above, we summarize schematically in Fig. 4d the preparation mechanism of the Cu₄O₃/CSBC composite. The two-step ball milling processes, BM-1 and BM-2, facilitate the uniform dispersion of refined copper formate particles onto the surface of CSBC. During the solid-state sintering process, copper formate underwent gradual cation reduction processes to generate Cu, which was then oxidized to Cu₂O by O₂ and further oxidized to CuO in air (Step 1 in Fig. 4d). The stoichiometric ratio of Cu₂O to CuO is crucial for the formation of high-purity Cu₄O₃^[9b]. CSBC plays a crucial role in regulating the ratio of Cu₂O and CuO in the reaction system: its weak reduction capacity could reduce CuO partially into Cu₂O but not all. Subsequently, the CuO and Cu₂O mixtures with an appropriate 2:1 ratio underwent the symproportionation reaction to form Cu₄O₃ (Step 2 in Fig. 4d). Meanwhile, the strong mechanical collisions of BM-2, air thermal decomposition, and oxidation-reduction reaction with CuO, increased the content of oxygen-containing functional groups, e.g., C = O and O-C = O, and reduced that of the C-H groups of CSBC.

2.3 Antibacterial property

The antibacterial properties of the Cu₄O₃/CSBC composite were investigated using Gram-negative bacterium Escherichia coli E. coli, Gram-positive bacterium Staphylococcus aureus S. aureus, and methicillin-resistant Staphylococcus aureus MRSA as bacterial models. MRSA is a significant cause of hospital and community-associated infections, characterized by its multidrug resistance.^[24] We evaluated the antibacterial efficacy of the Cu₄O₃/CSBC composite across various concentration gradients on three bacteria and determined the minimum inhibitory concentration (MIC) for each bacterium. Untreated CSBC and blank groups were applied as controls to evaluate the antibacterial activity of the Cu₄O₃/CSBC composite. As depicted in Fig. 5a, the antibacterial rate gradually declined from > 99.9% as the concentration of Cu₄O₃/CSBC composite decreased, indicating a positive correlation between the bactericidal effect and concentration. At the concentration of 5 µg mL^-1 for the Cu₄O₃/CSBC composite, E. coli was effectively eradicated and unable to proliferate on the nutrient medium. Notably, compared to the antibacterial effect against E. coli, the Cu₄O₃/CSBC composite showed lower MIC values against the Gram-positive bacteria S. aureus and MRSA: there were few surviving S. aureus or MRSA bacteria detected on the Petri dishes at concentrations of 0.100 µg mL^-1 and 0.125 µg mL^-1 of the Cu₄O₃/CSBC composite, respectively. The variation in the antibacterial efficacy of the Cu₄O₃/CSBC composite against Gram-negative and Gram-positive bacteria aligns with previously reported results regarding Cu₄O₃, potentially attributed to disparities in the cell membrane structure between the two bacterial strains.^[25] In contrast, both the CSBC and blank (Fig. 5a). These results indicate that the MIC values of the Cu₄O₃/CSBC composite against E. coli, S. aureus, and MRSA were 5 µg mL^-1, 0.100 µg mL^-1, and 0.125 µg mL^-1, respectively (Table S4, Supporting Information). Compared with the previous reports on the antibacterial properties of Cu₄O₃,^[9a] the antibacterial effect was enhanced by at least three orders of magnitude for the Cu₄O₃/CSBC composite in this work. Furthermore, we compared the MIC values of reported inorganic antibacterial materials against MRSA (Table 1). The Cu₄O₃/CSBC composite exhibits a significantly lower MIC value, indicating its efficient and notable antibacterial ability against superbug MRSA.

Furthermore, we applied SEM to observe the surface morphology of bacteria before and after treatment with the Cu₄O₃/CSBC composite (Fig. 5b). E. coli exhibited a rod-shaped morphology, while S. aureus and MRSA appeared spherical. Prior to the treatment, the bacteria displayed smooth surfaces with intact cell walls and membrane structures, but presented irregular morphologies with the wrinkling of the cell walls due to shrinkage after treated with the Cu₄O₃/CSBC composite.

Table 1

The MIC values comparison of inorganic antibacterial materials against *MRSA*.
Samples	MIC values for MRSA (µg mL^− 1)	Refs.
Cu₄O₃/CSBC composite	0.125	this work
Ag NPs (average size: 8.55–20.3 nm)	8.125	^[26]
Si-Ag	2500–5000	^[27]
Graphene oxide-Ag nanocomposite (average size: 9.4 ± 2.8 nm)	15	^[28]
Cu NPs (particle size: 50 nm)	1.875–3.75	^[29]
CuO NPs (particle size: 40 nm)	75–150	^[30]
Cu₂O@ZrP hybrid nanosheet	200	^[4c]
ZnO nanorods	23 ± 1.09	^[31]
ZnO NPs (average size: 20–25 nm)	125	^[32]
Heterogeneous TiO₂/ZnO Nanocomposite	80–2500	^[33]

2.4 Antibacterial mechanism

A number of studies have investigated the antibacterial mechanisms of CuO and Cu₂O, primarily focusing on the release of Cu ions, the induction and generation of reactive oxygen species (ROS), and direct contact with bacteria but few studies were carried out on Cu₄O₃.^[34] In order to investigate the antibacterial mechanism of the Cu₄O₃/CSBC composite, we first analyzed the release kinetics of Cu ions from the Cu₄O₃/CSBC composite by ICP-OES test. As shown in Fig. 6a, the cumulative release of Cu ions exhibited a positive correlation with immersion time during the 48-hour testing period. The cumulative release of Cu ions reached 2.71 ppm within 48 h, with the highest release rate of 235 ppb h^− 1 occurring in the initial 2 h (Figure S5, Supporting Information). The rapid release ensures the highly efficient antibacterial performance of the Cu₄O₃/CSBC composite. With prolonged immersion time, the Cu ions release rate decelerated gradually, which may be caused by the adsorption of copper ions by CSBC,^[35] indicating a slow-release process that ensures long-lasting antibacterial efficacy.

Both Cu(I) and Cu(II) ions have been reported to possess antibacterial effects and can induce the production of ROS by the redox reaction. Notably, the antibacterial efficacy of Cu(I) ions is significantly stronger than that of Cu(II) ions.^[36] Despite exhibiting a mixed valence of Cu(I) and Cu(II) at the Cu 2p XPS spectrum, the release mechanism of copper ions in Cu₄O₃ remains poorly understood. Initially, we detected the release of Cu(I) ions using bathocuproinedisulfonic acid disodium salt (BCS) as a specific chelator of Cu(I) ions. BCS can form stable complexes with Cu(I) ions, characterized by typical absorption peaks in the range of 450–500 nm.^[37] To minimize testing errors resulting from the oxidation of Cu(I) ions,^[38] we conducted an anaerobic condition for the solution system. As depicted in Fig. 6b, the control BCS exhibited no absorbance within the wavelength of 450–500 nm, while the absorbance of BCS treated with the Cu₄O₃/CSBC composite displays a characteristic absorption peak at 483 nm.^[37] The absorbance exhibited a gradual increment over the 48-hour test period, indicating a slow-release kinetics of Cu(I) ions within the Cu₄O₃/CSBC composite. Furthermore, we obtained the linear regression equation by measuring the standard curve between the absorbance of BCS at 483 nm and the concentration of Cu(I) ions (Figure S6, Supporting Information), thereby quantitatively evaluating the release of Cu(I) ions (Figure S7, Supporting Information). Then, the release concentration of Cu(II) ions was obtained by the difference between the total release concentration of Cu ions (ICP-OES results) and the release concentrations of Cu(I) ions. As can be seen in Figure S7 (Supporting Information), the cumulative release of Cu(II) ions also increased continuously during the 48-hour immersion experiment.

To further investigate the solvation mechanism of copper ions in Cu₄O₃, density functional theory (DFT) calculations were performed to compute the vacancy formation energy of Cu. Figure 6c illustrates the ball-and-stick model of Cu₄O₃ with body-centered tetragonal lattice structure (No.141, I4₁/amd space group), characterized by the built of inter-penetrating chains of Cu(I)-O and Cu(II)-O.^[39] The (101) surface model of Cu₄O₃ was constructed and optimized (Fig. 6d), containing three types of copper: monovalent copper (Cu-I), tetra-coordinated divalent copper (Cu-II-a), and tri-coordinated divalent copper (Cu-II-b). Subsequently, the vacancy formation energies of these copper types (Fig. 6e) were calculated using density functional theory (DFT). The calculation results (Table S5, Supporting Information) indicate that Cu-I exhibits the lowest vacancy formation energy, suggesting that Cu(I) ions are more easily dissolved than Cu-II-a and Cu-II-b ions. Additionally, the (100) surface model was constructed to calculate the vacancy formation energies of Cu-I and Cu-II (Fig. 6f). The results (Table S6, Supporting Information) demonstrate that the vacancy formation energy of Cu-I is lower than that of Cu-II again, consistent with the results of the (101) surface. These DFT calculations suggest that Cu(I) ions may dissolve more easily than Cu(II) ions from the surface of Cu₄O₃, and thus participate more efficiently in the antimicrobial process. The varied vacancy formation energies on the surfaces suggest the successive dissolution of Cu(I) followed by Cu(II) ions that may contribute to the long-lasting antimicrobial performance via the Cu-ion release mechanism.

Moreover, the stability of Cu₄O₃/CSBC composite in solution is critical. We confirmed the good chemical stability of the Cu₄O₃/CSBC composite by the 48-hour immersion in aqueous solution through comparison of XRD spectra (Fig. 6g). No obvious peaks were detected corresponding to Cu₂O, CuO, or Cu. Additionally, the intensity of Cu₄O₃ diffraction peaks gradually decreased with prolonged immersion time. We selected the strongest intensity of the XRD diffraction peak, (202), as the representative peak and compared it with the intensity of the amorphous diffraction peak of CSBC (I_d(202)/I_d(CSBC)). The comparison results (Table S7, Supporting Information) revealed that the ratio of I_d(202)/I_d(CSBC) decreased with increasing immersion time, further demonstrating the gradual release process of Cu ions from Cu₄O₃.

Subsequently, we assessed the generation of superoxide anion (\(\:\bullet\:{{\text{O}}_{2}}^{-}\)) using nitroblue tetrazolium (NBT) as reactant to evaluate the ROS generation capacity of the Cu₄O₃/CSBC composite.^[40] \(\:\bullet\:{{\text{O}}_{2}}^{-}\) can reduce NBT to water-insoluble formazan.^[41] Fig. 6h illustrates the absorbance changes of the NBT solution over 60 min after the addition of Cu₄O₃/CSBC composite. Compared to the NBT solution control, the intensity of the absorption peak at 259 nm significantly decreased after 10 min of adding the Cu₄O₃/CSBC composite, indicating the generation of \(\:\bullet\:{{\text{O}}_{2}}^{-}\).^[41] Over time, the absorbance of the NBT solution declined steadily, signifying the continuous degradation of NBT and a gradual rise in the concentration of \(\:\bullet\:{{\text{O}}_{2}}^{-}\). These above results indicate that the Cu₄O₃/CSBC composite can produce ROS and participate in the antibacterial mechanism.

Based on the above analysis, we summarize the antibacterial mechanism of the Cu₄O₃/CSBC composite as illustrated in Fig. 6i. The antibacterial efficacy of inorganic antibacterial agents is heavily influenced by their particle size, morphology, and dispersion.^[16] The Cu₄O₃/CSBC composite contains well-dispersed nano-sized Cu₄O₃, forming the foundation for their potent antibacterial activity and strong affinity for bacterial cells. Bacteria possess negatively charged cell surfaces, and the sustained slow release of Cu(I) and Cu(II) ions from the Cu₄O₃/CSBC composite can adhere to the bacterial surfaces, enhancing cell permeability and disrupting the cell walls/membrane structures to facilitate penetration of Cu ions into the cell interior. Concurrently, Cu(I) and Cu(II) ions, in conjunction with dissolved oxygen in water, participate in the production of ROS.^[36c] The disruption of the cell membrane facilitates the entry of more Cu ions and ROS into the cell interior. Within the cell, Cu ions further stimulate ROS generation, leading to DNA denaturation, lipid and protein structural damage, culminating in cell death.

3.1. Materials

Basic copper carbonate (CuCO₃·Cu(OH)₂, 54–56% Cu, pure), formic acid (99% pure), anhydrous zinc acetate (99.5% pure), and anhydrous ethanol (99.8% pure) were purchased from Shanghai Macklin Biochemical Co., Ltd. All reagents were used without further purification.

Corn straw, palm leaves, bagasse, and fir branches are used as raw materials for the preparation of biochar. Among them, corn straw was collected from farmland in Zibo, Shandong province, China; Palm leaves and fir branches were collected from the campus of Shanghai University, China; Bagasse comes from sugarcane purchased from a fruit store.

3.2. Bacteria

Bacterial strains Gram-negative Escherichia coli (ATCC 25922), Gram-positive Staphylococcus aureus (ATCC 6538), and Methicillin-resistant Staphylococcus aureus MRSA (ATCC 43300) were purchased from the American Type Culture Collection (ATCC, USA).

3.3. Materials characterizations

X-ray diffraction (XRD) spectrum was measured by the Bruker-D2 PHASE X-ray diffraction system (Cu Kα radiation, λ = 1.54184 Å, Bruker, Germany). The morphology and chemical compositions were analyzed by scanning electron microscopy (SEM, Sigma 500, Zeiss, Germany), transmission electron microscopy (TEM, accelerating voltage: 300 kV, FEI THEMIS ETEM G3, Thermo Fisher, US), and energy-dispersive X-ray spectrum (EDX) elemental mapping (80T, Oxford, UK), respectively. The surface chemical states of samples were measured by X-ray photoelectron spectroscopy (XPS, Al Kα source, Escalab Xi+, Thermo Fisher, US), and the whole data was corrected by C 1s value (284.8 eV). The structural composition of the Cu₄O₃/CSBC composite was examined using Fourier-transform infrared spectroscopy (FTIR, Nicolet iS50, Thermo Fisher Scientific, US). The content of Cu was measured by inductively coupled plasma-optical emission spectrometry (ICP-OES, 730 Series, Agilent Technologies, US). The Cu content of the sample was calculated from the element analysis obtained via ICP-OES. The morphology of bacteria was observed by SEM (NOVA NanoSEM 450, FEI, US). The absorbance of the samples was analyzed by Ultraviolet-visible (UV-vis) spectra (UV-2450, Shimadzu, Japan).

3.4. Methods

3.4.1. Preparation of the Cu₄O₃/biochar composite

Preparation of biochar and copper formate

Corn stover biochar (CSBC) was prepared by slow pyrolysis method in an Ar atmosphere.^[10b] Corn stover underwent cutting and pulverization, followed by sequential washing three times with deionized water and anhydrous ethanol separately to eliminate potential ionic impurities. Subsequently, the powder was dried at 70 ℃ for 2 h. The dried corn stover powder was placed into a quartz crucible and then transferred into a tube furnace. After post-Ar displacement, the furnace was heated to 300 ℃ at a heating rate of 10 ℃ min^− 1 under Ar atmosphere and maintained for 2 hours. Upon cooling to room temperature, the sample was retrieved from the furnace to obtain CSBC.

Copper formate was prepared by precipitation reaction using basic copper carbonate and 30% formic acid aqueous solution.^[16]

Ball milling process (BM-1 and BM-2)

BM-1: The BM-1 process for copper formate has followed the method outlined in our previous work.^[16] In brief, copper formate (20 g), zirconiagrinding balls (60 g), and anhydrous ethanol (20 mL) were added to 2 zirconia jars, and then placed in a planetary ball mill (MIQI, QM3SP2, Changsha, China) and milled for12 h at 300 rpm. After milling, the milled mixture was dried in a drying chamber at 70 ℃ for 6 h and then sieved by a stainless mesh.

BM-2: Pre-set preparation of Cu₄O₃/CSBC composite with a Cu₄O₃ mass ratio of 15 wt.%. The ball-milled copper formate (152.3 mg) and CSBC powders (425 mg) were added equally to 2 zirconia jars. Each jar was supplemented with grinding balls (12 g) and anhydrous ethanol (2.0 mL). Subsequently, the jars were transferred to a planetary ball mill, and subjected to ball milling for 1.5 h at 300 rpm. Following the ball milling process, the jars were placed in a drying chamber at 60 ℃ for 2 h to remove ethanol. Finally, the resulting copper formate/CSBC precursor powder was sieved by a stainless mesh screen.

Solid-state sintering process

Copper formate/CSBC precursor (20 mg) was put in a quartz crucible and transferred to the isothermal region of a box-type resistance furnace, the furnace was heated to 230 ℃ with a heating rate of 10 ℃ min^− 1 and maintained for 12 min. The Cu₄O₃/CSBC composite was finally obtained after air cooling to room temperature.

3.4.2. Quenching reactions

The box-type resistance furnace was heated to 230 ℃ and then waited for 10 min until the furnace temperature stabilized. The six quartz crucibles containing pre-prepared copper formate/CSBC precursor were put into the isothermal region rapidly and timing started immediately after the temperature stabilized at 230 ℃. When sintered at 230 ℃ for 2 min, one sample was immediately taken out by a crucible tongs, covered with aluminum foil, and placed in a dewar filled with liquid nitrogen for cooling (40 s). Similarly, the remaining 5 samples were taken out after sintering at 230 ℃ for 4 min, 6 min, 8 min, 10 min, and 12 min, respectively, and were quenched by rapid cooling with liquid nitrogen.

3.4.3. Determination of antibacterial properties

To assess the antibacterial properties of the Cu₄O₃/CSBC composite, Gram-negative Escherichia coli E. coli, Gram-positive Staphylococcus aureus S. aureus, and Methicillin-resistant Staphylococcus aureus MRSA were employed as model bacteria. The minimum inhibitory concentration (MIC) was tested using the oscillation method in accordance with the national standard of China "GB/T 21510 − 2008". Nutrient agar medium, nutrient broth, phosphate buffer solution (PBS, 0.03 mol L^− 1), Petri dishes, conical flasks, test tubes, pipette tips, and filter paper were sterilized in an autoclave for 20 min at 121 ℃ and 103 kPa. Subsequently, nutrient agar medium (15 mL) was evenly distributed into a 90 mm diameter Petri dish and allowed to solidify at room temperature. E. coli, S. aureus, and MRSA were each inoculated into nutrient broth (100 mL) and incubated at 37 ℃ and 130 rpm for 18 h with shaking to achieve an initial inoculum suspension with a viable cell count of 10⁸ CFU mL^− 1. Following incubation, the bacterial solution was diluted to 10³ CFU mL^− 1 using nutrient broth and PBS buffer solution.

The bacterial solution, PBS buffer solution, and various concentrations of the Cu₄O₃/CSBC composite solution were mixed in a volume ratio of 1:4:5. Following a 4-h incubation period at 37 ℃ and 150 rpm with shaking, the treated mixture (5 µL) was dropped onto a nutrient agar medium plate. Subsequently, the growth of bacteria was assessed following a 24-h incubation period at 37 ℃. The lowest concentration at which bacteria cease growing represents the MIC of the test sample. CSBC was employed as a control for the antibacterial test, with the tested concentration matching that of CSBC content (85 wt.%) in the Cu₄O₃/CSBC composite.

The three bacteria, E. coli, S. aureus, and MRSA, were sampled before and after the antibacterial test treatment, fixed with glutaraldehyde, and dehydrated in a gradient with anhydrous ethanol. The morphology of the bacteria before and after treatment with the Cu₄O₃/CSBC composite was observed by SEM.

3.4.4. Determination of release of copper ions

The release of Cu(I) ions was detected using bathocuproinedisulfonic acid disodium salt (BCS). The Cu₄O₃/CSBC composite (5 mg) was put into a double-necked round-bottomed flask containing BCS solution (20 mL) with a concentration of 0.5 mM. Inert gas displacement was performed sequentially by vacuum pump evacuation and nitrogen filling and circulated three times to exclude oxygen from the solution system. The suspension was then sonicated for 2 min and then stirred at 500 rpm and timed, the upper suspension (200 µL) was aspirated at 30 min, 1 h, 2 h, 6 h, 12 h, and 24 h, respectively. The recovered suspension was centrifuged and filtered through a 0.11 µm filter membrane to remove insoluble substances. UV-Vis spectroscopy was employed to measure the absorbance of the samples in the wavelength range of λ = 190–700 nm.

3.4.5. ROS test

The reactive oxygen species (ROS) generation capability of Cu₄O₃/CSBC composite was assessed using nitroblue tetrazolium (NBT) to detect superoxide anion (\(\:\bullet\:{{\text{O}}_{2}}^{-}\)). The Cu₄O₃/CSBC composite (5 mg) was placed into a beaker containing NBT solution (20 mL) with a concentration of 25 µM. The suspension was then ultrasonicated for 2 min followed by stirring at 500 rpm, with timing initiated upon stirring commencement. At intervals of 10 min, 20 min, 30 min, and 60 min, the upper suspension (200 µL) was aspirated, underwent centrifugation and filtration through a 0.11 µm filter membrane to eliminate insoluble substances. UV-Vis spectroscopy was utilized to measure the absorbance of the samples in the wavelength range of λ = 190–500 nm to assess changes in NBT concentration.

3.4.6. Computational methods

All density functional theory (DFT) calculations were performed by using the Vienna ab initio simulations package (VASP).^[42] The interaction between valence electrons and ion cores is described by the projector augmented wave (PAW) method,^[43], and plane waves with a kinetic energy cutoff of 520 eV were used for expanding electron wave functions. The exchange-correlation interaction is treated by the Perdew-Burke-Ernzerhof (PBE) functional within the generalized gradient approximation (GGA).^[44] The integration over the first Brillouin zone is performed on a uniformed k-point mesh with a grid density of ~ 2π × 0.02 Å⁻¹ along each periodic direction, that is 9×9×9 for bulk Cu₄O₃ and 4×8×1 for the slab models. The Hubbard-U correction with parameters U = 7.5 eV and J = 0.98 eV is adopted for the on-site repulsion of Cu-3d orbitals, which is widely used in Cu-O systems.^[45]

In this study, the Cu₄O₃/biochar composite with superior antimicrobial properties was developed through the solid-state synthesis strategy of ball milling and sintering processes. This in-situ preparation method demonstrates the characteristics of simplicity, efficiency, eco-friendliness, and universality of the biochar source, achieving the high-purity and stable support of Cu₄O₃. The preparation mechanism of Cu₄O₃/CSBC composite was investigated, in which copper formate was used as the copper source, while biochar serves as the reducing agent, support carrier, and dispersant within the preparation and reaction system, playing a crucial role in regulating the stoichiometric ratio of Cu₂O and CuO. This preparation method achieved the first solid-state symproportionation reaction of copper oxides beyond liquid-phase methods.

The antibacterial test shows that the Cu₄O₃/CSBC composite exhibited highly efficient bactericidal effects against E. coli, S. aureus, and MRSA, particularly demonstrating significantly low MIC value against S. aureus and superbug MRSA. Furthermore, we analyzed the antibacterial mechanism of the Cu₄O₃/CSBC composite through experiment and DFT computational approaches, revealing the dual effects from both the slow successive release of Cu(I)/Cu(II) ions and the generation of ROS. The Cu₄O₃/CSBC composite is expected to be applied to public healthcare to enhance protection against pathogenic microbial infections, including superbugs. This solid-state synthesis approach prepares high-purity Cu₄O₃/CSBC composite via symproportionation reactions by combining ball milling and sintering processes and is anticipated to achieve large-scale preparation and broaden the application scope of Cu₄O₃.

Conflict of Interest

The authors declare no conflict of interest.

Acknowledgements

We thank the financial supports of the National Natural Science Foundation of China (Nos. 52373227, 52201016, and 91641128) and the National Key R&D Program of China (Nos. 2017YFB0701502 and 2017YFB0702901). This work was also supported by the Shanghai Technical Service Center for Advanced Ceramics Structure Design and Precision Manufacturing (No. 20DZ2294000), and the Shanghai Technical Service Center of Science and Engineering Computing, Shanghai University. The authors acknowledge the Beijing Super Cloud Computing Center, Hefei Advanced Computing Center, and Shanghai University for providing HPC resources.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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The authors declare no competing interests.

Cu4O3SKCEJSI.docx

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Green solid-state synthesis of Cu₄O₃/biochar composites with high antimicrobial activity

Status:

Version 1

Abstract

Figures

1. Introduction

2. Results and Discussion

2.1 Preparation and characterization of the Cu₄O₃/biochar composite

2.2 Formation mechanism of the Cu₄O₃/CSBC composite

2.3 Antibacterial property

2.4 Antibacterial mechanism

3. Materials and methods

3.1. Materials

3.2. Bacteria

3.3. Materials characterizations

3.4. Methods

3.4.1. Preparation of the Cu₄O₃/biochar composite

3.4.2. Quenching reactions

3.4.3. Determination of antibacterial properties

3.4.4. Determination of release of copper ions

3.4.5. ROS test

3.4.6. Computational methods

4. Conclusion

Declarations

Conflict of Interest

Acknowledgements

Data Availability Statement

References

Additional Declarations

Supplementary Files

Status:

Version 1

Green solid-state synthesis of Cu4O3/biochar composites with high antimicrobial activity

Status:

Version 1

Abstract

Figures

1. Introduction

2. Results and Discussion

2.1 Preparation and characterization of the Cu4O3/biochar composite

2.2 Formation mechanism of the Cu4O3/CSBC composite

2.3 Antibacterial property

2.4 Antibacterial mechanism

3. Materials and methods

3.1. Materials

3.2. Bacteria

3.3. Materials characterizations

3.4. Methods

3.4.1. Preparation of the Cu4O3/biochar composite

3.4.2. Quenching reactions

3.4.3. Determination of antibacterial properties

3.4.4. Determination of release of copper ions

3.4.5. ROS test

3.4.6. Computational methods

4. Conclusion

Declarations

Conflict of Interest

Acknowledgements

Data Availability Statement

References

Additional Declarations

Supplementary Files

Status:

Version 1

Green solid-state synthesis of Cu₄O₃/biochar composites with high antimicrobial activity

2.1 Preparation and characterization of the Cu₄O₃/biochar composite

2.2 Formation mechanism of the Cu₄O₃/CSBC composite

3.4.1. Preparation of the Cu₄O₃/biochar composite