Constructing supported Ag antibacterial nano-agent with high activity and stability for broad-spectrum antibiosis

Analytical grade chemicals including Co(NO₃)₂·6H₂O, Al(NO₃)₃·9H₂O, AgNO₃, NaOH, and Na₂CO₃ were supplied by Aladdin reagent Co, Ltd and used without further purification. Deionized water was used in the all experiments.

The Co₂Al-CO₃ ²⁻-LDHs nanoplates were prepared by the hydrothermal method reported previously [39]. A solution of Co(NO₃)₂·6H₂O (0.008 mol) and Al(NO₃)₃·9H₂O (0.004 mol) in 40 ml of water was quickly added to 40 ml of NaOH (0.025 mol) and Na₂CO₃ (0.1 g) solution with vigorous agitation at room temperature. The mixture was transferred into an autoclave, followed by hydrothermal treatment at 80 °C for 5 h. The precipitate was separated by centrifugation and washed thoroughly with water.

LDHs-supported Ag nanoparticles (Ag/CoAl-LDHs) were prepared by a facile in situ oxidation-reduction process between the CoAl-LDHs nanoplate and the AgNO₃ solution. In a typical procedure, the suspension of CoAl-LDHs nanoplates (about 4 mg ml⁻¹) was firstly obtained by fresh LDHs precipitate ultrasonically dispersed in the deionized water. Afterwards, AgNO₃ solution (12 ml, 5 mg ml⁻¹) was added into LDHs suspensions (50 ml, 4 mg ml⁻¹) at 50 °C for 6 h under stirring. The redox reaction process resulted in the in situ deposition of Ag nanoparticles onto the surface of CoAl-LDHs nanoplate. Finally, the resulting product was separated by centrifugation, washed thoroughly with water and dried under vacuum for 24 h.

The zeta potential and dynamic lighting scatting (DLS) diameter were measured by photon correlation spectroscopy (PCS, Nanosizer Nano ZS, MALVERN Instruments). TEM images were obtained by a JEM-2100F high resolution transmission electron microscope with an accelerating voltage of 200 kV. Powder x-ray diffraction (XRD) patterns were collected on a Bruker x-ray diffractometer (D8 focus, Cu Kα, λ = 0.151 78 nm) operated at a scan rate of 0.1° s⁻¹. Fourier transform infrared spectra (FT-IR) were acquired on a Varian Excalibur 3100 FTIR spectrophotometer using the KBr pellet technique in the range 4000−500 cm⁻¹. Inductively coupled plasma atomic emission spectroscopy (ICP-AES) data were obtained on a Varian 710−OES. XPS measurements used an ESCALAB 250Xi (Thermo Scientific), equipped with a non-monochromatized Al-Kα x-ray source (hν = 1486.7 eV).

To evaluate antimicrobial activity, the antimicrobial tests of Ag/CoAl-LDHs with varied concentrations (50, 100, 500, and 1000 μg ml⁻¹) were performed by using Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) as a microorganism, respectively. The microbial population was analyzed by counting colony-forming units (CFUs) using the pour-plate technique. S. aureus or E. coli was first incubated with varied concentrations of Ag/CoAl-LDHs at 37 °C for 30 min in PBS. A 100 ml portion of the S. aureus or E. coli suspension was spread on a solid LB (Luria Bertani medium) nutrient agar plate. The colonies formed after 16 h incubation at 37 °C for S. aureus or E. coli were counted. The diameter of the solid agar plates was 90 mm. In addition, the antimicrobial activity of CoAl-LDHs (1000 μg ml⁻¹) and only PBS as two control samples were evaluated under same method, respectively. All tests were performed in triplicate.

To study the antibacterial efficiency and stability, the antibacterial growth curves of S. aureus and E. coli treated with Ag/CoAl-LDHs (1000 μg ml⁻¹) was investigated using Bioscreen C. The two strains in the logarithmic growth phase were suspended in nutrient broth medium at 10⁻⁶ CFU/ml. Bacterial suspension (30 μl) was mixed with 180 μl nutrient broth medium and 30 μl of Ag/CoAl-LDHs, such that the final concentrations of Ag/CoAl-LDHs were 1000 μg ml⁻¹. The same volume of physiological saline was added instead of the Ag/CoAl-LDHs solution in the negative control. The medium was incubated at 37 °C for 33 h in the Bioscreen C system (Oy Growth Curves, Helsinki, Finland). All tests were performed in triplicate.

LDHs-supported Ag antibacterial nanomaterials (Ag/CoAl-LDHs) were synthetized by the in situ immobilization of the Ag NPs onto the CoAl-LDHs surface (see the Experimental section for details). The CoAl-LDHs precursors with Co/Al ratio of ∼2 was firstly synthetized on the basis of the hydrothermal method reported previously by our group. The XRD patterns of the CoAl-LDHs precursors (figure 1(A-a)) showed a series of diffraction peaks at 11.7°, 23.5°, 34.6°, 60.4°, and 61.7°, which can be assigned to (003), (006), (009), (110), and (113) characteristic reflections of the LDH structure [39]. The basal spacing (d₀₀₃) for CoAl-LDHs precursors was 0.769 nm, which was in agreement with the value reported for CoAl-CO₃ ²⁻-LDHs phase [39]. In addition, (110) and (113) reflections can be observed, indicating the formation of a well crystallized CoAl-LDHs phase [39]. Figure 1(B) showed that the CoAl-LDHs precursors had a mean lateral size of ∼165 nm, as determined by Dynamic Lighting Scatting (DLS). As shown in TEM image (figure 1(C)), the CoAl-LDHs precursors had a typical morphology of two-dimensional nanoplate, and the nanoplates thickness of ∼6 nm can be observed from the cross section of CoAl-LDHs (in inset).

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After an in situ deposition process, LDHs-supported Ag antibacterial nanomaterials were obtained (see the Experimental section for details). Compared with the CoAl-LDHs precursors, the obtained Ag/CoAl-LDHs (figure 1(A-b)) showed a similar characteristic reflection of LDHs phase, and a series of extra diffraction peaks at 38.1°, 44.4°, and 64.4° were observed, which can be assigned to metallic Ag phase (JCPDS 04-0783). This implied the formation of Ag/CoAl-LDHs antibacterial nanomaterials. TEM and HRTEM were further applied to confirm the morphology and fine structure of Ag/CoAl-LDHs (figures 1(D)–(F)). Figure 1(D) showed that Ag/CoAl-LDHs inherited the nanoplate morphology of CoAl-LDHs precursors, and with close average size of LDHs nanoplate. Notably, it can be observed that high contrast black dots (figures 1(D) and (E)) with a uniform size distribution (16.5 nm) were well-dispersed on the surface of CoAl-LDHs nanoplates, suggesting the deposition of Ag nanoparticles owing to the higher bright-field image contrast compared with CoAl-LDHs nanoplates. To study the crystalline structure of LDHs-supported Ag nanoparticles, the HRTEM image for one single Ag nanoparticle was shown in figure 1(F). The lattice fringe of 0.235 nm corresponded to the (111) plane of the face centered cubic (fcc) Ag phase [40]. These results revealed that Ag nanoparticles were successfully in situ loaded onto the surface of CoAl-LDHs surface, forming Ag/CoAl-LDHs antibacterial nanomaterials. X-ray photoelectron spectroscopy (XPS) was performed to investigate the surface electronic structure of Ag/CoAl-LDHs (figures 2(A) and (B)). Previous investigations of XPS spectra for Ag 3d_5/2 reported that binding energy values of 367.4 and 367.8 eV corresponded to AgO and Ag₂O, respectively, while that of metallic Ag are usually located around 368.2 eV [41, 42]. In this work, as shown in the figure 2(A), the Ag 3d_5/2 XPS peak for Ag/CoAl-LDHs at ∼368.2 eV was observed, which can be assigned the Ag⁰ state. Moreover, one O1s peak around 531.9 eV, corresponding to surface adsorption oxygen species (e.g., adsorbed hydroxyl, adsorbed carbonate) can be observed in Ag/CoAl-LDHs, while the characteristic peaks of silver oxidized species (around 529.5 eV) were not found (figure 2(B)) [42–44]. Therefore, these indicated that the surface electronic states of supported Ag nanoparticles in Ag/CoAl-LDHs belonged to the zero-valence state of metallic Ag. In addition, the loading amount of Ag nanoparticles in CoAl-LDHs was observed to be ∼15 wt% by ICP−AES spectroscopy.

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The in situ loading of Ag nanoparticles onto the surface of CoAl-LDHs nanoplate can be understood from the viewpoint of oxidation-reduction process: there was a redox reaction between Co²⁺ in LDHs layers and Ag⁺ from AgNO₃ solution, inducing the in situ reducing and dispersing of the Ag nanoparticles onto the LDHs surface. As shown in XPS of Co 2P (figure 2(C)), the peaks for CoAl-LDHs at ∼797.5, ∼781.3, ∼803.5, and ∼787.1 eV were assigned to the Co²⁺ state and their two satellite peaks [42, 44]. Notably, after an in situ deposition process, the two Co 2P peaks for Ag/CoAl-LDHs shifted to low binding energy (796.8 and 780.6 eV, respectively) compared with that of CoAl-LDHs, indicating the occurrence of a partial valence change from Co²⁺ to Co³⁺. Meanwhile, after an in situ reduction process, Al 2P peak (around 74.3 eV) in Ag/CoAl-LDHs did not shift compared with that of CoAl-LDHs [42, 44]. These revealed that the driving force for the immobilization of the Ag nanoparticles originated from the redox reaction between Ag⁺ from AgNO₃ solution and Co²⁺ in LDHs layers. Thus, it was reasonable to propose that CoAl-LDHs nanoplates played the roles of both a support and a reductant without any external agent, and subsequently the redox reaction between Ag⁺ and Co²⁺ led to the in situ spontaneous deposition of Ag nanoparticles onto the LDH surface. In addition, the specific ordered arrangement of the Co²⁺ and Al³⁺ cations in the LDH layers provided well-dispersive and uniform Co²⁺ active sites for the reduction of Ag⁺ from AgNO₃ solution, imparting a good microenvironment for the nucleation and stabilization of the Ag nanoparticles onto the LDHs surface. Therefore, this work provides a green and facile synthetic approach for supported Ag antibacterial nanomaterials with a uniform size and high dispersion of Ag nanoparticles.

To evaluate antimicrobial activity, the antimicrobial tests for Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) was performed by Ag/CoAl-LDHs with varied concentrations (50, 100, 500, and 1000 μg ml⁻¹), respectively, based on counting colony-forming units (CFUs) method. In the case of S. aureus (figure 3(A)), many bacterial colonies flourished in the control plate. However, when co-incubated with Ag/CoAl-LDHs antibacterial nanomaterials, the bacterial cells of S. aureus decreased gradually with the treatment concentration increasing from 50 to 1000 μg ml⁻¹. Correspondingly, antibacterial rate for S. aureus (calculated by CFUs method) increased from 24% to 73% with the treatment concentration (figure 3(B)). These results indicated that the antibacterial ability of Ag/CoAl-LDHs for S. aureus enhanced with the treatment concentration. Moreover, under the same treatment concentration (1000 μg ml⁻¹), Ag/CoAl-LDHs showed a stronger bacteriostatic effect compared with that of CoAl-LDHs (73% versus 57%). For E. coli (figure 3(A)), no obvious antibacterial effect was observed for the control group, and Ag/CoAl-LDHs at concentration of 50 μg ml⁻¹ (figure 3(C)) showed a high colony counts but low antibacterial rate (14%). In contrast, when the treatment concentration was higher than 100 μg ml⁻¹ (figures 3(A) and (C)), the colony counts decreased significantly, and the antibacterial rate remained at a high level (about 74% to 78%), suggesting a strong antibacterial ability for E. coli. Additionally, Ag/CoAl-LDHs showed much better bacteriostatic effect than CoAl-LDHs (74% versus 20%), under the same treatment concentration (1000 μg ml⁻¹). To study the antibacterial efficiency and stability, the antibacterial growth curves of S. aureus and E. coli treated with Ag/CoAl-LDHs (1000 μg ml⁻¹) was further investigated using Bioscreen C (figures 3(D) and (E)). It was worth noting that the growth of S. aureus and E. coli in the groups treated with Ag/CoAl-LDHs were essentially suppressed within 5 h, respectively, and no regained growth was found at all subsequent investigated time points. Moreover, antibacterial rate for S. aureus and E. coli, calculated by the antibacterial growth curves, were close to those based on CFUs method. Therefore, antibacterial experiments verified that Ag/CoAl-LDHs as antibacterial nanomaterials not only had an efficient antibacterial activity against both S. aureus and E. coli, but also exhibited a very stable antibacterial properties for both bacteria.

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