Molybdenum disulfide (MoS₂) layered bulk crystals, n-Butyllithium (2.5 M in cyclohexane) were purchased from Sigma Aldrich (United States). Polyvinylpyrrolidone (PVP) with the average MW 58000 was purchased from Aladdin Reagent Company (China). AgNO₃ was obtained from Macklin. Ag nanoparticles were successfully grown on MoS₂ nanosheets by 300 W xenon lamp (PLS-SXE100) irradiation. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) was provided from Sigma Biochemical Technology Co., Ltd. (Shanghai, China). LIVE/DEAD™ Baclight™ Bacterial Viability Kit (L7012) was purchased from Invitrogen (China). Cell counting Kit-8 (CCK-8) was purchased from Beyotime (China). Staphylococcus aureus and L929 cells were supplied by the Henan Eye Institute.

Scanning electron Microscope (SEM) images were captured Field Launch Scanning Electron Microscope (Zeiss Sigma 500, Germany). Transmission electron microscope (TEM) images taken on by field emission transmission electron microscope (JEM-2100F, Japan). The diameter of particles and zeta potential of MoS₂/Ag NSs were detected by Malvern ZEM 3700 equipment. The antibacterial property of samples with MTT was investigated by 2,104 Multilabel Microplate Reader (PerkinElmer). UV-Vis-NIR spectra were detected with a spectrophotometer from Agilent Technologies (Cary 5,000). Fluorescence pictures of microbial were observed with a Nikon 80i fluorescence microscope.

The similar approach outlined in the prior research was used to prepare the MoS₂ nanosheets (Chen et al., 2022b). The specific steps were as follows: the grinded MoS₂ crystals (100 mg) were immersed in a solution of n-butyllithium (2.5 M in cyclohexane, 5 mL) and kept in glove boxes for 48 h to obtain lithium intercalation compounds. The precipitate at the bottom was washed three times with hexane after taking of the upper layer of n-butyllithium, followed by adding 50 mL of water and sonicated for 30 min to produce a homogeneous suspension. The large-size nanosheets were removed by centrifuging at 5,000 rpm for 10 min. The supernatant was further centrifugation products at 5,000–12000 rpm were collected and washed three times with DI water to obtain MoS₂ nanosheets.

40 mg PVP was added to 20 mL of synthesized MoS₂ suspension in triplicate, and the AgNO₃ (1 mg/mL) solution with various volumes (0.5, 1, and 2 mL) was added to the mixture. After stirring evenly, the mixture was lit under a xenon lamp at 300 W for 30 min. The crude products with different silver content were denoted as MoS₂/Ag1, MoS₂/Ag2, and MoS₂/Ag3 and were washed two times with DI water to get the MoS₂/Ag nanosheets with different silver contents.

Using the Cell Counting Kit-8, the cytotoxicity of MoS₂/Ag3 NSs was determined (CCK-8, Beyotime). Each well plate was injected with 7 to 8 10³ L929 cells for 24 h in a thermostatic incubator set to 37°C with 5% CO₂, and then the wells were filled with 100 μL of the MoS₂/Ag3 NSs solution for another 24 h. Subsequently, a fresh medium (100 μL) containing 10% CCK-8 agentia was added. This medium was then cultured for 4 h in a thermostatic incubator. Enzyme labeling (PerkinElmer Envision, England) was used to detect the optical density and cell viability at 450 nm.

Staphylococcus aureus (S. aureus) was used as the bacterial model. Frozen strains were first resuspended and transferred to columbia blood agar plates to be incubated overnight in a 37°C incubator, and then took a single colony by inoculation loops and transferred it to 4 mL LB medium and incubated for 12 h at 220 rpm on a shaker. Bacterial suspension was diluted to 10⁷ CFU/mL for inhibition experiments. Microdilution, plate counting, fluorescence staining, and scanning electron microscopy were used to examine the antibacterial activity and mechanism of MoS₂/Ag nanosheets against S. aureus.

The synthesis process of MoS₂/Ag nanosheets was schematically stated in Scheme 1. The MoS₂ nanosheets were fabricated and obtained according the previous procedure (Chen et al., 2022b). To load the silver nanoparticles (Ag NPs) onto the surface of MoS₂ nanosheets, AgNO₃ was first mixed with MoS₂ nanosheets solution and then reduced under the irradiation of a Xenon lamp, the highly dispersed Ag NPs could be obtained. The scanning electron microscope (SEM) images clearly showed that the MoS₂/Ag3 NSs exhibited the uniform nanosheet morphology with a size around 200–400 nm ( Figures 1A, B ). The TEM images disclosed that the Ag NPs were successfully dispersed on the surface of MoS₂ nanosheets (Figure 1C), and its high-resolution transmission electron microscopy (HRTEM) image (Figure 1D) further presented that the lattice fringes of 0.27 and 0.15 nm belonged to planes of MoS₂ (100) and Ag (220), respectively, indicating Ag NPs were successfully loaded onto the surface of nanosheets. Promisingly, the selected area electron diffraction (SAED) pattern of MoS₂/Ag3 NSs was characterized by the presence of bright diffraction spots with regular hexagon (inset of Figure 1D), signifying its single-crystalline nature.

Both the MoS₂ and MoS₂/Ag3 NSs were further characterized by X-ray photoelectron spectroscopy (XPS). The XPS survey spectra revealed the presence of six elements (C, N, O, S, Mo, and Ag) in MoS₂/Ag3 NSs (Figure 2A), while the MoS₂ nanosheets were found to lack the Ag element (Supplementary Figure S1A). Compared with the XPS Ag 3d spectrum of MoS₂ nanosheets (Supplementary Figure S1B), that of MoS₂/Ag3 NSs exhibited two distinct peaks at 372.98 eV and 366.98 eV corresponding to Ag 3d_3/2 and Ag 3d_5/2 of Ag (0) (Figure 2B) (Qiao et al., 2017; Li et al., 2022a), which indicated the fabrication of MoS₂/Ag NSs. As shown in Figure 2C, the high-resolution XPS Mo 3d spectrum of the MoS₂/Ag3 NSs given two main peaks at 230.68 eV and 227.48 eV, which belonged to Mo 3d_3/2 and Mo 3d_5/2 of Mo (IV), respectively (Chen et al., 2022a). The characteristic peaks at 161.38 eV and 160.48 eV (Figure 2D) originated from S 2p_1/2 and S 2p_3/2 of S (II) (Zhou et al., 2020), respectively. The UV-Vis absorption spectroscopy data (Supplementary Figure S2) were used to confirm that Ag NPs had been synthesized without structural alteration of MoS₂. Moreover, it was found that the augmentation of silver content had negligible effect on particle size and Zeta potential of MoS₂/Ag3 NSs (Supplementary Figure S3).

As shown in Figure 3A, we evaluated the cytotoxicity of MoS₂/Ag NSs with the highest silver content (MoS₂/Ag3) by CCK-8 experiment in L929 cells. The viability of L929 cells was still higher than 85% after the treatment with MoS₂/Ag3 at the concentration of 50 μg/mL, showing that excellent biocompatibility and the great potential for in vivo and in vitro antibacterial applications.

Considering its great photodynamic performance and promising peroxidase-like ability (Supplementary Figure S4), the germproof capacity against S. aureus was further appraised by plate counting method. After co-incubation with the designed materials, it was found that the group of H₂O₂ or laser irradiation had a negligible antimicrobial effect against S. aureus (Figure 3B). Therefore, the optimal inhibitory concentration of all samples was determined under laser irradiation at the presence of H₂O₂. As presented in Figure 3B, the visual colony dramatically decreased with increasing concentrations of antibacterial agents, which showed the fascinating concentration-dependent bactericidal capacity. It was noting that the antibacterial effect was further enhanced with increasing density of Ag NPs (30 μg/mL), implying that maybe more ROS were activated to grievously destroy the morphology of bacteria. The antibacterial properties of MoS₂ NSs were also determined in terms of MTT assay. The results demonstrated that only a survival rate of 0.12% was performed in the group of MoS₂/Ag3 with the concentration of 30 μg/mL, but obvious colonies were observed in other groups (Figures 3B, C). Interestingly, an impressive bactericidal effect against S. aureus was observed when the concentration of MoS₂/Ag nanosheets was raised.

In order to intuitively observe the inhibitory effect against S. aureus under irradiation of laser (660 nm) or in the presence of H₂O₂, we kept up with the plate-counting antibacterial experiment with MoS₂/Ag nanosheets at the concentration of 30 μg/mL. It has been reported that H₂O₂ (100 μM) together with near-infrared irradiation exhibited hardly inhibitory effect (Wei et al., 2021). Although the growth of microorganisms was not completely suppressed, H₂O₂ or laser irradiation had a more conspicuous antibacterial effect than only material in other groups (Figure 3D), suggesting that the ROS was insufficient to kill S. aureus at the current concentration. Following the combination of laser irradiation and H₂O₂ therapy, S. aureus survival rate continued to decline, indicating synergistically antibacterial capacity with regard to photodynamic therapy and peroxidase-like strategy (Li et al., 2020; Zhang et al., 2022a). Compared to the other groups, MoS₂/Ag3 exhibited the most robust antimicrobial ability and almost all bacteria were inactivated, demonstrating the antibacterial effectiveness might be improved with the addition of Ag. Thus, it could be speculated that the practical bactericidal process under irradiation was dominated by the synergistic impact of MoS₂ and Ag NPs to boost ROS formation.

MoS₂/Ag3 NSs (30 μg/mL) was utilized to carry out the live-dead bacteria staining experiment for the sake of investigating the antibacterial mechanism. All S. aureus could be stained by green fluorescence (SYTO™-9) and only damaged S. aureus presented red fluorescence (PI). Fluorescence images showed (Supplementary Figure S5) that membranes of S. aureus would not be destroyed in terms of free H₂O₂, NIR irradiation (10 min), or combination of H₂O₂ and NIR irradiation (10 min). Since the red fluorescence in the H₂O₂ condition only was practically identical to that of a single laser irradiation in Figure 4A, so it is almost harmless to conduct independent action. But the peroxidase-like activity of MoS₂/Ag3 NSs could kill off large numbers of bacteria in the presence of H₂O₂, implying that the synergistic effect made S. aureus unable to survive.

To further appraise the antimicrobic capacity of MoS₂/Ag3 NSs, surface morphologies of antibacterial with different treatment were detected by SEM. It was observed that the bacteria of the PBS group maintained a smooth and unbroken membrane, meanwhile the S. aureus treated by H₂O₂ or NIR revealed no evident difference with the PBS group, suggesting the intact structure of S. aureus (Figure 4B). However, obvious wrinkles and destruction of S. aureus were observed in the MoS₂/Ag3, MoS₂/Ag3 + H₂O₂, and MoS₂/Ag3 + NIR groups (Figure 4B). Promisingly, the bacterial membranes shrunk more seriously and even destroyed (signaled by yellow arrows) in the MoS₂/Ag3 + H₂O₂ + NIR group (Figure 4B), which attributed to the ample ROS synergistically generated by the catalysis of MoS₂/Ag3 under the NIR laser irradiation in the presence of H₂O₂.

Inspired by the results of the in vitro antibacterial assays described above, we hypothesized a multi-level synergistic antibacterial mechanism ( Figure 5 ). The sterilization mechanism of Ag NPs was mainly to puncture the cell membrane of their tiny size (Akter et al., 2018). When irradiated by a 660 nm laser, the valence band electrons of MoS₂ were stimulated to change into the conduction band and produce electron-hole pairs (Zhu et al., 2020). Additionally, combining Ag NPs and MoS₂ could facilitate electron transport and prevent the compounding of electron-hole pairs, generating a significant amount of photoelectrons and holes (Ma et al., 2016). Virtually, the positively charged holes had a robust oxidability and could yield ¹O₂ when reacted with oxygen (Karkhanechi et al., 2014; Zhang et al., 2020; Zhao et al., 2022a; Luo et al., 2022). Importantly, H₂O₂ could be catalyzed to produce ROS for the sake of the peroxidase-like ability of MoS₂ (Wang et al., 2016). As a result, when bacteria were exposed to near-infrared laser, the synergistic action assaulted the bacterial membrane, causing the bacterial metabolic barrier and ultimately causing bacterial mortality.