2D Ag Ion-Loaded Anionic Nanosheets for Polymer-Based Film with Durable Antibacterial Activities.

Silver (Ag) has been demonstrated to have excellent performance to kill bacteria, fungi, and some viruses because it can release positively charged Ag ions with highly antibacterial and antifungal activities. However, effectively controlling the slow release of Ag ions is the key to preparing high-performance Ag-based antibacterial agents, which remains a challenge. In this work, we have developed a new Ag-based antibacterial agent composed of Ag ions loaded on 2D anionic 2D Sb3P2O14 3- nanosheets (denoted as Ag-Sb3P2O14). 2D anionic nanosheets not only adsorb a large amount of Ag ions but also control their slow release through electrostatic interaction between nanosheets and Ag ions. 2D Ag-Sb3P2O14 nanofillers enable excellent high antibacterial activities for the poly(vinylidene fluoride) (PVDF) film composites against microorganisms including Escherichia coli and Staphylococcus aureus. Moreover, the PVDF membrane with 5 wt % 2D Ag-Sb3P2O14 nanofillers can kill almost all bacterial after 50 times washing, demonstrating its excellent durable antibacterial activities. This work opens up a new and promising route to durable Ag-based antibacterial agents for polymer-based composites.

Introduction

Poly(vinylidene fluoride) (PVDF) has gained wide applications as membrane and film materials owing to its unique physicochemical properties such as high thermal stability, high mechanical strength, and excellent membrane formation.[1−3] Such properties make PVDF a good candidate to prepare polymer-based antibacterial membranes and films for applications in packaging materials, textiles, and water treatment.[3,4] However, previous studies demonstrated that pure PVDF membrane is easily adsorbed by various biocontaminants including bacteria. The adsorbed bacteria are able to multiply and clone to form a sticky biofilm on the PVDF membranes and films, which can further adsorb more contaminants.

Recently, much effort has been devoted to develop PVDF-based films with excellent antibacterial property to prevent the accumulation of the surface contaminants. Various organic and inorganic fillers have been reported to incorporate in the PVDF membrane to increase its antibacterial, such as Ag nanoparticles,[5,6] functional graphene,[7−9] MoO3 nanowires,[10] nano ZnO powder,[11] TiO2 nanoparticles,[12,13] and metal organic frameworks.[14,15] Comparted to other metal and metal oxides, Ag has demonstrated excellent antibacterial and antifungal activities due to its high cytotoxicity against a wide variety of microorganisms.[16] Although the antibacterial mechanism of Ag is not clear,[17] recent studies have demonstrated that the Ag ions released from the Ag metal are responsible for the antibacterial activities.[18] For example, the released Ag ion interacts with the sulfhydryl groups from the enzymes and proteins of the cell membrane,[19] leading to protein deactivation and bacterial death. The antibacterial effect of the Ag metal is dependent on the concentration of the released Ag ion around the bacteria, which means that ultrasmall Ag nanoparticles have antibacterial property, much better than bulk Ag and micro-sized Ag particles because of their high surface area. However, the Ag nanoparticles are easy to agglomerate when they are separated from aqueous solutions. In order to prevent the aggregation of ultrasmall Ag nanoparticles in the solid state, they are usually anchored on other inorganic nanoparticles, nanowires, and 2D nanosheets with a high surface area,[20−22] which can significantly improve the antibacterial activities.

Apart from achieving Ag ions from the isolated Ag nanoparticles, silver salts can release Ag ions to kill microorganisms. Generally, insoluble silver salt powder releases Ag ions too slowly and soluble silver salt powder releases Ag ions too fast, which renders them not suitable as antibacterial agents. In order to control the slow release of Ag ions, they are fixed at the organic polymer matrix and inorganic nanoparticles by chelation and adsorption.[23−25] For instance, Fe3O4 nanoparticles with functional groups were reported to adsorb the Ag ions, which can serve as an antibacterial agent to enhance the antibacterial performance of carrageenan-based packing films.[26] However, the loading of Ag ions on the nanoparticle is not high owing to the low surface area. In principle, the adsorption amount and sustained release of Ag ions are dependent on the surface area of substrates and the interaction between the Ag ion and substrates. Comparted to nanoparticles, 2D ultrathin nanosheets with a large aspect ratio and a high surface area have potential as a candidate to load more Ag ions. Moreover, the electrostatic adsorption has a stronger interaction force than simple physical adsorption, which not only increases the adsorption amount but also reduces the release rate of Ag ions. Therefore, it is reasonable to assume that the negatively charged 2D nanosheets are an excellent adsorption candidate to achieve a high-performance Ag ion-based antibacterial agent.

Herein, we report a novel antibacterial agent composed of Ag ions adsorbed on 2D Sb3P2O143– nanosheets (denoted as Ag-Sb3P2O14), which can significantly improve the durable antibacterial activities of the PVDF membrane. 2D Sb3P2O143– nanosheets show graphene-like nanostructures with a high surface area and a strong negative charge and can adsorb large amounts of Ag ions and control their slow release. As a result, the PVDF film with 5% Ag-Sb3P2O14 exhibits excellent antibacterial activities against Escherichia coli and Staphylococcus aureus and has a good antibacterial effect even after 50 times washing.

Experimental Section

Materials

KNO3 (Adamas, 99%), Sb2O3 (Adamas, 99%), NH4H2PO4 (Adamas, 99.99%), HNO3 (diluted, 65–68 wt %), and AgNO3(Adamas, 99.8%) were purchased from Sinopharm Chemical.

Preparation of 2D H3Sb3P2O14 Nanosheets

2D H3Sb3P2O14 nanosheets could be synthesized based on previous literature.[27,28] First, 2.66 g of KNO3, 5.06 g of Sb2O3, and 3.51 g of NH4H2PO4 were mixed and heated up to obtain K3Sb3P2O14 powder. After that, 4 g of K3Sb3P2O14 powder was treated with 500 mL of nitric acid (8 M) for 12 h, which was repeated three times to complete the proton exchange reaction and provide H3Sb3P2O14 bulks, followed by exfoliation via vigorous stirring in a pure water solution. Finally, the suspension was centrifuged under 3000 rpm for 30 min, and the non-exfoliated bulk material was removed to obtain ultrathin 2D H3Sb3P2O14 nanosheets.

Preparation of Ag-Sb3P2O14 Nanosheets

Ag-Sb3P2O14 nanosheet powder was synthesized by dissolving AgNO3 in a colloidal suspension of H3Sb3P2O14 in water (0.26 wt %). Once the dispersed H3Sb3P2O14 nanosheets were contacted by the dissolved AgNO3, a flocculation phenomenon occurred immediately because the negatively charged Sb3P2O143– attracts the positive Ag+ to form Ag-Sb3P2O14.The Ag-Sb3P2O14 nanosheet powder was collected by filtration, washed with deionized water, and dried at 80 °C.

Preparation of Ag-Sb3P2O14/PVDF Thin Film

The Ag-Sb3P2O14/PVDF thin film was prepared by casting the slurry of PVDF (Mw = 1000000) mixed with 5, 10, 15, and 20 wt % of Ag-Sb3P2O14 powder on the glass pane. The pure PVDF film and the PVDF composite film with 10 wt % H3Sb3P2O14 were prepared as a contrast group. Finally, these materials were collected after being dried at 80 °C for 8 h.

Characterization of Ag-Sb3P2O14/PVDF

The surface morphologies of the samples were conducted by scanning electron microscopy (SEM, JSM-7500F) combined with energy-dispersive X-ray spectroscopy (EDS, JEOL, JSM-7610F), transmission electron microscopy (TEM, JEOL 2100F), and optical imaging (SHOT ON MI 6X). The structural properties and chemical composition analysis for samples were performed using X-ray diffraction (XRD, Rigaku D/Max-2200V PC) and X-ray photoelectron spectroscopy (XPS, Thermo Scientific K-Alpha).

Antibacterial Activities

Escherichia coli (E. coli, ATCC25922) and Staphylococcus aureus (S. aureus, ATCC6538) served as model bacteria for evaluating the antibacterial activities of the Ag-Sb3P2O14/PVDF film.

Zone of inhibition test was applied to qualitatively measure the antibacterial activities of samples. First, the bacterial suspension was grown in Luria-Bertani (LB) agar medium at 37 °C for 24 h. Then, the suspension was diluted to a density of approximately 5 × 107 CFU/mL by mixing with culture medium, followed by uniformly spreading 100 μL of diluted suspension over the agar plates. As for examining the inhibition zone, the samples were put on the center of agar plates and cultured at 37 °C for 24 h. Finally, a digital camera was utilized to directly record the inhibition zone.

As for the bacterial inhibition rate test, the bacterial suspension was diluted to a density of approximately 1 × 108 CFU/mL, and then the suspension was centrifuged for 3 min × 3 times with PBS solution. 0.075 g of Ag-Sb3P2O14/PVDF thin film was then introduced into the bacterial suspension (7 × 106 CFU/mL) and incubated at 37 °C. Besides, the culture medium without bacteria served as the blank controlled sample, and the bacteria suspension without examined samples served as the negative controlled sample. After culturing for 3, 6, and 24 h, the bacterial suspension was diluted 10 times and spread on the LB plate. The number of bacteria was determined by the colony counting method.

The bacterial inhibition rate was calculated based on the following equation: bacterial inhibition rate (%) = (B – A)/B × 100; the antibacterial activity (R) was calculated by the equation: R = lg A – lg B, where A and B were pointed out to be the optical density of the negative controlled samples and tested samples, respectively. The release concentration for the Ag+ from the Ag-Sb3P2O14/PVDF films was quantitatively measured by inductively coupled plasma-optical emission spectroscopy (Agilent 720ES).

Stability and Durability Testing

Antibacterial membrane washing method of Shanghai University was used to test the washing durability of the Ag-Sb3P2O4/PVDF film. The total liquid volume is 50 mL, including standard detergent ECE 2 g/L and 10 steel balls. Then, the Ag-Sb3P2O4/PVDF thin film was put in it and kept at 40 °C for 45 min, washed twice with water at 40 °C (1 min each time), and finally dried.

Results and Discussion

2D Sb3P2O143– nanosheets play an important role in adsorbing the Ag ions and controlling the slow release of Ag ions, which was synthesized through a “top-down” strategy,[27] as shown in Figure a. First, bulk K3Sb3P2O14 with a layer crystal structure was prepared by a conventional solid-phase method. SEM image shows the particle-like morphology with microsize (Figure b), and the enlarged SEM image reveals the layer-stacked structure of the big particles, which is similar to that of bulk MXenes (Figure c). The XRD pattern confirms the K3Sb3P2O14 phase because the main diffraction peaks are indexed to K3Sb3P2O14 (JCPDS 78-1030), as shown in Figure d. Afterward, 3D H3Sb3P2O14 bulks can be obtained by immersing the bulk K3Sb3P2O14 into nitric acid, followed by centrifugation and exfoliation. The XRD result also confirmed the successful transition from K3Sb3P2O14 to H3Sb3P2O14, indicating the successful ion-exchange reaction (Figure g). The obtained H3Sb3P2O14 bulk was further exfoliated, and the desired 2D H3Sb3P2O14 nanosheets were obtained through simple mechanical agitation in aqueous solution, which was confirmed by the SEM and TEM images. As displayed in Figure e, the SEM image shows the micro-sized sheetlike nanostructures. TEM further reveals its 2D graphene-like nanosheet morphology (Figure f).

Figure 1

(a) Schematic of the synthesis strategy of H3Sb3P2O14. (b) Low magnification top-view SEM image and (c) enlarged image of K3Sb3P2O14. (d) XRD pattern of K3Sb3P2O14. (e) SEM and (f) TEM images of H3Sb3P2O14. (g) XRD pattern of H3Sb3P2O14.

The 2D H3Sb3P2O14 nanosheets are easily and uniformly dispersed in H2O to form a homogeneous and transparent suspension (Figure a). In aqueous solution, 2D H3Sb3P2O14 nanosheets are easy to form 2D Sb3P2O143– anionic nanosheets with a rich negatively charge via ionization.[27] Because of strong electrostatic repulsions between nanosheets, the colloidal suspension of 2D H3Sb3P2O14 nanosheets is very stable. In order to obtain Ag-Sb3P2O14 nanosheets, AgNO3 was added to the colloidal suspension, where the positively charged Ag+ are attracted by the negatively charged nanosheets by electrostatic interactions (Figure a,b). The SEM with the corresponding EDS mapping images of the nanosheets verify that the Ag ions have been adsorbed on the 2D Sb3P2O143– nanosheets. As shown in Figure c, the surface of nanosheets becomes rough. The EDS mapping shows the uniform distribution of Ag, N, O, P, and Sb elements. The presence of N element is attributed to NO3– adsorption. The EDS spectrum shows that the mass loading of Ag is as high as 33.4 wt %, indicating high adsorption of Ag ions (Figure c). Furthermore, the XRD pattern of Ag-Sb3P2O14 shows that all main diffraction peaks are indexed to the phase of H3Sb3P2O14 (JCPDS 40-0218), indicating that the adsorbed Ag ion compound is amorphous (Figure d). Besides, XPS spectra shows a strong Ag 3d peak, indicating the presence of Ag in the Ag-Sb3P2O14 nanosheets. The corresponding high-resolution peaks were well fitted to the Ag d3/2 orbital (373.68 eV) and the Ag d5/2 orbital (367.68 eV), further confirming that Ag (I) existed in the Ag-Sb3P2O14 nanosheets rather than metal Ag (0) (Figure e,f).[29] Compared with AgNO3 chelated by poly(2-ethyl-2-oxazoline), the lower binding energy of valence electrons of Ag in Ag-Sb3P2O14 nanosheets demonstrated the weaker interaction between the silver cation and the substrate, which is beneficial to the release of Ag ions.[29] Moreover, the TEM image reveals that the nanoparticle-like Ag+ compound including NO3– (AgNO3) is uniformly dispersed on the nanosheets in the form of nanoparticles, which is consistent with the EDS results (Figure g).

Figure 2

(a) Preparation of Ag-Sb3P2O14 powder. (b) Schematic diagram of Ag-Sb3P2O14 nanosheets. (c) Top-view SEM image and EDS map obtained from the Ag-Sb3P2O14 powder. (d) XRD pattern of Ag-Sb3P2O14 powder. (e,f) XPS spectra of Ag-Sb3P2O14 powder: (e) survey scan and (f) Ag 3d. (g) TEM image of Ag-Sb3P2O14 powder.

To improve the antibacterial activity of the PVDF film, Ag-Sb3P2O14 served as the nanofiller to prepare the PVDF composite film by the way of solution blending, as shown in Figure a. SEM was used to investigate the morphology of the PVDF film with different loadings of Ag-Sb3P2O14 nanofillers (Figure b–g). Obviously, pure PVDF membrane displays a flat and dense surface without obvious pores or undulation (Figure c). After incorporating 5 wt % Ag-Sb3P2O14, the surface became a little rough, and no big aggregate of Ag-Sb3P2O14 was found, indicating its good dispersion (Figure d). It was found that the surface roughness of the composite membrane increased with the increase of Ag-Sb3P2O14. With a very high loading of Ag-Sb3P2O14 as high as 20 wt %, some Ag-Sb3P2O14 aggregates were detected because of the high loading of fillers. For comparison, the PVDF membrane with 10 wt % H3Sb3P2O14 nanosheets was prepared, showing a rough surface (Figure b). It should be noted that no H3Sb3P2O14 aggregates were detected with the loading of 10 wt % H3Sb3P2O14 nanosheets.

Figure 3

(a) Schematic of the synthesis strategy of Ag-Sb3P2O14/PVDF film. Top-view SEM images of the (b) PVDF film with 10 wt % H3Sb3P2O14 film, (c) pure PVDF film, and PVD film with (d) 5 wt % Ag-Sb3P2O14, (e) 10 wt % Ag-Sb3P2O14, (f) 15 wt % Ag-Sb3P2O14, and (g) 20 wt % Ag-Sb3P2O14.

In order to evaluate the antibacterial activities of the Ag-Sb3P2O14/PVDF film, the zone of inhibition tests were conducted first, where the diameter of the round films was 0.02 m. As displayed in Figure a, pure PVDF films did not show any inhibition zone for both E. coli and S. aureus, indicating that the pure PVDF film cannot kill the bacterial around the film. In contrast, it could be observed that the PVDF composite samples with Ag-Sb3P2O14 nanosheets demonstrated antibacterial effect for both E. coli and S. aureus, confirming the important role of the Ag-Sb3P2O14 nanosheets on the antibacterial performance. In detail, with the increasing of the content of Ag-Sb3P2O14 in the films, the diameter of the corresponding inhibition zones increases (Figure b,c). The diameter is as high as 2.7 mm for E. coli and 1.5 mm for S. aureus for the 20 wt % Ag-Sb3P2O14/PVDF film, proving a strong connection between the Ag content and the antibacterial activity. It should be noted that the diameter of the inhibition zone is not high, indicating the slow release of Ag ions from the Ag-Sb3P2O14 nanosheets.

Figure 4

(a–c) Inhibition zone of (b) E. coli and (c) S. aureus of pure PVDF, 10 wt % H3Sb3P2O14, 5 wt % Ag-Sb3P2O14/PVDF, 10 wt % Ag-Sb3P2O14/PVDF, 15 wt % Ag-Sb3P2O14/PVDF, and 20 wt % Ag-Sb3P2O14/PVDF.

Moreover, the standard plate count method was applied to quantitatively assess the antibacterial activity for the above Ag-Sb3P2O14/PVDF film. The results of the E. coli and S. aureus colonies with 7 × 106 CFU/mL concentration on the scaffolds have been displayed in Figure a. Obviously, the pure PVDF and 10 wt % H3Sb3P2O14/PVDF samples still maintained tons of bacteria on the surface of samples, expressing extremely low bacterial activities for both E. coli and S. aureus. However, after mixing the Ag-Sb3P2O14 nanosheets into the composite film, the bacterial inhibition performance was hugely enhanced. All PVDF films with Ag-Sb3P2O14 showed over 99.99% antibacterial rate (Figure b,c), which may be attributed to the excellent antibacterial activity of Ag-Sb3P2O14 nanosheets.

Figure 5

(a–c) Antibacterial rate of (b) E. coli and (c) S. aureus of PVDF films with 10 wt % H3Sb3P2O14, 5 wt % Ag-Sb3P2O14/PVDF, 10 wt % Ag-Sb3P2O14/PVDF, 15 wt % Ag-Sb3P2O14/PVDF, and 20 wt % Ag-Sb3P2O14/PVDF.

The Ag+ release evaluation of Ag-Sb3P2O14 nanosheets was carried out by measuring the concentration of Ag ions as a function of rest time, as displayed in Figure a. Initially, 0.1 mg/mL Ag-Sb3P2O14 nanosheets were suspended in the water solution and then placed for monitoring the concentration of Ag ions. After 3 days, the above solution has a Ag+ concentration as high as 0.54 mg/L, indicating that the Ag-Sb3P2O14 nanosheets can effectively release the Ag ions, which is higher than the concentration (from 0.1 to 10 μg/mL) the bacteria could live and could effectively inhibit the bacterial growth and reproduction.[30] This also explains that the films with a low loading of Ag-Sb3P2O14 shows excellent antibacterial activities (Figure c). Remarkably, with the prolongation of rest time, the Ag+ cumulative concentration gradually and slowly increases. The concentration of Ag ions is about 0.65 mg/L after placing for 5 days and is up to 0.90 mg/L after 7 days, verifying the excellent ability to slowly release Ag ions and ensuring the long-term antibacterial performance. Moreover, in order to detect the durable antibacterial performance of Ag-Sb3P2O14 nanosheets, the antibacterial activity of the PVDF film with 5 wt % Ag-Sb3P2O14 was examined after different washing times (Figure b). As shown in Figure b, the composite film exhibited a high antibacterial performance and kills almost all E. coli and S. aureus even after washing 50 times, confirming its durable antibacterial activity of the PVDF based films. The excellent durable antibacterial effect of Ag-Sb3P2O14/PVDF film against E. coli and S. aureus is attributed the ability of the slow release of Ag ions for the Ag-Sb3P2O14 nanosheets.

Figure 6

(a) Release concentration of Ag ions of PVDF with 5 wt % Ag-Sb3P2O14. (b) Antimicrobial activities against E. coli and S. aureus of the PVDF film with 5 wt % Ag-Sb3P2O14 after different washing times. (c) Proposed mechanism schematic of Ag-Sb3P2O14/PVDF films.

Conclusions

In summary, we have designed and prepared a new Ag-based antibacterial agent, which is composed of 2D Ag-Sb3P2O14 nanosheets. 2D anionic nanosheets can load large amounts of Ag ions on the surface through electrostatic interactions, which also control the slow release of surface Ag ions in aqueous solution. Inductively coupled plasma–optical emission spectrometry results reveal that the concentration of Ag ions of the solution containing 2D Ag-Sb3P2O14 nanosheets increase with an increase of the soaking time, exhibiting outstanding continuous sterilization performance. The as-prepared 2D Ag-Sb3P2O14 nanofillers enable the PVDF membrane with high antibacterial activities against both E. coli and S. aureus (over 99.99% antibacterial rate). Besides, it should be noted that the antibacterial ability of the PVDF film with 5 wt % 2D Ag-Sb3P2O14 nanosheets remains high antibacterial effect after 50 times washing, further confirming the slow release of Ag ions anchored on the 2D anionic nanosheets. Such distinguished antibacterial activities of the Ag-Sb3P2O14/PVDF film indicate the application potential to develop polymer-based composites with durable antimicrobial activities against microorganisms.