Introduction

Bacteria-caused diseases threaten the human health seriously and have turned to be the leading causes of death worldwide [1, 2]. The abuse of antibiotics has led to the increase in drug-resistant bacteria, which makes the battle against bacterial infections even worse [3]. In this context, developing new and effective antibacterial agents is imminent.

Nanomaterials with antimicrobial activities have garnered many attentions both in scientific researches and in industrial applications during the past decades. Comparing with the small molecular antibiotics, antimicrobial nanomaterials generally display better performances in terms of long-lasting antibacterial activities and environmental toxicities. Besides, antimicrobial nanomaterials circumvent the emergences of drug-resistant bacteria, which is a serious problem for the small molecular antibiotics because of their specific targets of action [4]. Recently, post-graphene 2D materials (pg-2DMs), like transition metal dichalcogenides (TMDs), black phosphorus (BP) and layered double hydroxides (LDHs), have been presented as promising candidates to combat pathogens [5, 6]. The antibacterial mechanisms of pg-2DMs have been proposed as well, including (1) physical interactions (lipid extraction, “nano-knife” effect, etc.) that will damage cell membrane and block the cell material exchange, (2) photothermal effects and (3) photocatalytic generation of reactive oxygen species (ROS) [5].

The promising potentials of graphene in antimicrobial applications have greatly stimulated the interest of researchers in fabricating various post-graphene 2D materials-based antimicrobial agents (pg-2DMs-AA) (Fig. 1a). Thus far, many pg-2DMs, such as transition metal dichalcogenides (TMDs) [7, 8], black phosphorus (BP) [9, 10] and layered double hydroxides (LDHs) [11], MXenes [12, 13], g-C3N4 [14, 15] and In2Se3 [16], have been utilized to prepare antimicrobial agents (Fig. 1b). In this review, we overview the methods for enhancing the antimicrobial activities of pg-2DMs-AA and the biosafety assessment of the pg-2DMs-AA.

Figure 1
figure 1

a Numbers of pg-2DMs-AA-associated publications; b percentages of the pg-2DMs that have been used to prepare antimicrobial agents

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Strategies to improve the antimicrobial activities of post-graphene 2D materials-based antimicrobial agents

Synthesizing new-generation antibacterial agents based on pg-2DMs is of great interest to scientists and engineers in many fields such as biomedicine and food packing. Due to their natures, pristine pg-2DMs generally display limited antimicrobial activities. Thus, to fabricate efficient antibacterial agents, the first and crucial step is to choose rational strategies to boost the antimicrobial capacities of pg-2DMs-AA. To summarize these strategies, herein we conduct a comprehensive literature survey by inputting keywords (i.e., 2D materials, two-dimensional materials, MoS2, black phosphorus, g-C3N4, antibacterial, antimicrobial, disinfection, sterilization) into the widely used databases (e.g., Web of Science, Scopus, Google Scholar). Notably, we do not intend to summarize all the methods employed in preparing pg-2DMs-AA, but only to introduce the frequently utilized and applicable approaches (Fig. 2).

Figure 2
figure 2

Schematic illustration of how the different strategies enhance the antimicrobial activities of pg-2DMs-AA. Strategy 1: fabricating single- or few-layer pg-2DMs; strategy 2: building vertical structures; strategies 3 and 4: decorating with other materials to enhance the photocatalytic and/or photothermal activity; strategy 5: loading with other antibacterial agents

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Fabricating single- or few-layer pg-2DMs

The researches about graphene-like 2D materials are greatly motivated by the unique physicochemical properties of these materials with the reduction in the thickness to single or few layers. In the antimicrobial field, reducing the layers of graphene significantly enhances its antimicrobial activities [17, 18]. In addition to graphene, other 2D materials also show the similar phenomenon [13, 19]. For instance, Yang et al. utilized the Li intercalation approach to prepare the chemically exfoliated few-layer MoS2 (ce-MoS2). Compared with the raw MoS2 powder, ce-MoS2 demonstrated a much stronger inactivation ability on Escherichia coli DH5α (Fig. 3a) [19]. The possible underlying mechanism is the decrease in the 2D materials’ layer will generate more atomically sharp edges, more active sites, larger surface to volume ratio and higher photothermal conversion efficiency (PCE), all of which can induce membrane and oxidative stress to microbes [19]. For some pg-2DMs, such as MoS2 and MoSe2, decreasing their layer numbers will also change their band gaps from indirect to direct band gaps [20, 21], which results in the increase in their photocatalytic and/or photothermal activities, and thereby improve their antimicrobial efficiencies. Now, the common approaches to prepare single- or few-layer pg-2DMs for antimicrobial applications include the top-down exfoliation method (e.g., ball milling exfoliation, liquid-based ultrasonic exfoliation) and the down-top method (e.g., hydro/solvo-thermal synthesis) (Fig. 2).

Figure 3
figure 3

a Viabilities of the E. coli DH5a incubated with different concentrations (5–80 μg/mL) of ce-MoS2 sheets or raw MoS2 powders for 2 h. Reproduced with permission from Ref. [19]. Copyright 2014, Royal Society of Chemistry. b Antibacterial properties of WX2-ssDNA and GO nanosheets. (The viabilities of E. coli cells decreased when they were treated with 80 μg/mL WX2-ssDNA or GO nanosheets for 5 h.) Reproduced with permission from Ref. [34]. Copyright 2016, American Chemical Society. c Fluorescence images of B. subtilis that were treated with 100 μg/mL MoS2, MoS2/rGO, MoS2/MXene, MnO2 and MnO2/GO nanomaterials for 3 h in dark. Red and green dots represented the live and dead bacteria, respectively. Reproduced with permission from Ref. [46]. Copyright 2018, American Chemical Society

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Ball milling exfoliation

The micromechanical cleavage technique is the traditional mechanical exfoliation method utilized to cleave thin graphene flakes from their parent bulk crystals by the scotch tape [22]. The method was soon applied in other 2D materials such as MoS2 and WSe2 [23]. However, this method is low yield and the thickness, size and shape of the resultant materials are difficult to control as this method is mainly operated by hands. Obviously, this method is for fundamental researches but not suitable for antimicrobial applications that need convenient and scalable productions of thin-layer 2D materials. To overcome this drawback, the method of ball milling, generating two types of force-shear force and compression force, has garnered attentions. For instance, Feng et al. mixed bulk MoS2 with chitosan and ionic liquid, which were ground in a planet ball mill afterward. After the grinding process, the mixture was collected and washed with acetone, N, N-dimethylformamide and 0.5% acetic for three times to remove the ionic liquid and the excess chitosan. Chitosan improved the dispersibility and stability of MoS2 and increased the thickness of the resultant MoS2 nanosheets (approximately 4.7 nm). This chitosan-modified MoS2 nanosheets inactivated E. coli and Staphylococcus aureus significantly [24]. Zhang et al. [25] exfoliated bulk MoS2 with the assistance of ionic liquid using a similar mechanical method, in which the authors used an agate mortar and a pestle to grind the mixture instead of the planet ball mill.

Liquid-based ultrasonic exfoliation methods

Ultrasonication is frequently used to exfoliate 2D materials as well. When the ultrasonic waves propagate through the medium, the interaction between the ultrasonic waves and the medium causes physicochemical changes, resulting in a series of chemical, thermal, electromagnetic and mechanical ultrasonic effects. The cavitation in the liquid is the main cause of exfoliation. When the cavitation bubble explodes, microjets and shock waves are generated, which lead to the erosion and structural damages on the material surfaces, as well as the exfoliation and collision between the material particles [26]. Yet, using ultrasonication to directly exfoliate 2D materials can hardly be accomplished in water unless some appropriate additives or stabilizers are supplemented, due to the strong hydrophobic characters of many 2D materials [27]. Organic solvents (e.g., isopropyl alcohol [28], dimethylformamide [29] and sodium cholate [30]) with appropriate surface tensions to match the surface energy of the targeted 2D material well, are often chosen because of their important roles in reducing the potential energy barriers that exist in the interlayers of bulk 2D materials and in the stabilization of resultant nanosheets by interfacial interactions [27]. Adding intercalants such as Li ions also improves the exfoliation of bulk 2D materials [19], because the insertion enlarges the interlayer spacings of bulk 2D materials and then weakens the interlayer interactions, and finally facilitates the subsequent ultrasonic exfoliation [27].

The aforementioned methods have the advantages of high yield and large scale, and the products also show good antibacterial activities. But some solvents, such as N-methyl-2-pyrrolidone and N-cyclohexyl-2-pyrrolidone, may adsorb on the resultant nanosheets and are difficult to be removed completely [31]. This is not conducive for the wider biomedical applications of the resultant materials. In addition, most of the used surfactants are synthetic, which will raise the concerns about the cost, environmental impact and biocompatibility of the resultant 2D materials [32]. Meanwhile, the alkali metal intercalation-assisted exfoliation is time-consuming (about several days) and the used organometallic materials are highly explosive and sensitive to the moisture and oxygen, which needs strict experimental conditions with extreme cautions [33]. To overcome these limitations, researchers pay attentions to the biomolecules (e.g., nucleic acids, proteins, polysaccharides) in recent years, as they can serve as the more sustainable and environment-friendly natural stabilizers for 2D materials [32]. Until now, nucleic acids [34], proteins [35] and chitosan [36] have been utilized successfully. For instance, Bang et al. have reported an effective and high-yield exfoliation technique for WS2 and WSe2 under aqueous conditions with the assistance of ssDNA. The nonpolar nucleobase of ssDNA can readily adsorb onto the surface of WS2 and WSe2, whereas the polar phosphate group is able to extend into and strongly interact with the aqueous medium, furnishing the resultant materials with colloidal stability due to the steric and electrostatic repulsion. Interestingly, the as-prepared WS2 and WSe2 nanosheets showed higher antibacterial activities against E. coli than that of graphene oxide (Fig. 3b) [34]. Silk fibroin also has an amphiphilic structure and hydrophilic/hydrophobic multi-block domains and thereby can be used for the exfoliation and stabilization of 2D materials. Huang et al. exfoliated bulk MoSe2 with the assistance of carboxyl-modified silk fibroin (CMSF). The authors report that the resultant MoSe2/CMSF nanosheets own peroxidase-like activity and can turn low concentrations of H2O2 into ROS to kill E. coli and Bacillus subtilis [35].

However, in all the above cases, exfoliated 2D materials are not covalently functionalized, which causes instability over time and limit their applications. Recently, Chou and coworkers have reported that chemically exfoliated MoS2 nanosheets with defects in both internal edges and perimeter edges are amenable to the thiol ligand modifications [37]. Similarly, Pandit et al. exfoliated MoS2 using the lithium intercalation-assisted sonication method. Then to functionalize the surface of the material, they mixed the exfoliated MoS2 nanosheets with different thiol ligand solutions. They found that the functionalized MoS2 nanosheets showed higher stability in aqueous media and the positively charged MoS2 nanosheets inactivated methicillin-resistant S. aureus and Pseudomonas aeruginosa effectively at very low concentrations (156 ppb and 78 ppb, respectively) [38]. This thiol ligand-based functionalization method is further optimized by Karunakaran et al. [39]. They synthesized surfactant thiol ligands with amphiphilic properties, by which they efficiently exfoliated and functionalized 2H-MoS2 simultaneously in aqueous medium without the assistance of lithium intercalation. The functionalized MoS2 nanosheets showed perfect bactericidal effects. All these works demonstrate the possibilities of fabricating pg-2DMs-AA with long-lasting stabilities by the covalent functionalization method.

Hydro-/solvo-thermal synthesis

The hydro- or solvo-thermal synthesis method, belonging to the down-top approaches, relies on the suitable “metal–organic” molecules as the precursors for the direct synthesis of the few-layer 2D materials in the solution [40]. The solvent contains the polymer or surfactant, and the precursor is kept in sealed vessels or autoclaves and heated over its boiling point. This process will promote the reduction of the precursor salts into 2D sheets. Because of large-scale production, easy and inexpensive synthesis of nanostructured materials, hydro-/solvo-thermal synthesis methods have been frequently used to attain few-layer 2D materials with interesting structures and sometimes the doping of foreign nanomaterials, which can endow excellent antimicrobial activities to the as-prepared materials. For instance, Cao et al. synthesized PEG-MoS2 nanosheets using a modified solvo-thermal method. In short, 60 mg thioacetamide (C2H5NS) and 30 mg sodium molybdate (Na2MoO4·2H2O) were dissolved in 20 mL PEG-200 aqueous solution (50%, v/v) and then transferred into a 100-mL polyphenylene-lined stainless steel autoclave, which was heated at 200 °C for 24 h afterward. The resultant MoS2-PEG nanosheets were further decorated with l-cysteine, silver ion and the cationic polyelectrolyte to obtain the PDDA-Ag+-Cys-MoS2 nanocomposites. This nanocomposite exhibited perfect broad-spectrum antibacterial activities due to its high accessibility of released Ag+ to the bacterial cell wall [41].

Building vertical structures

Past studies have shown that vertical graphene can kill bacteria via membrane destroy and ROS damage [42,43,44]. For instance, Lu and coworkers found that the antibacterial activity of graphene oxide was related to its orientation. Among the vertical, random and planar graphene oxide (GO), vertical structure exhibited the highest antibacterial activity against E. coli. The cell mortality of vertical GO (44.0 ± 8.7%) was 1.5 times higher than that of the random (25.7 ± 3.5%) and twice of the planer (19.2 ± 5.1%) [43]. Similar to graphene, MoS2 with vertical structures exhibits excellent bactericidal activity as well. For example, Liu et al. prepared the few-layered vertically aligned MoS2 (FLV-MoS2) by a chemical vapor deposition (CVD) technique. Under the visible light, FLV-MoS2 showed > 99.999% E. coli inactivation in 120 min. FLV-MoS2 in the dark showed < 50% disinfection efficiency after 120 min, and bulk MoS2 just showed a 54% efficiency over 120 min. The authors conclude that FLV-MoS2 owns a higher in-plane electrical conductivity and catalytic activity on the edge sites. Thus, FLV-MoS2 has a better electron–hole transport from MoS2 to the electrolytes and so exhibits a better photocatalytic activity, which increases the ROS generation and the subsequent bactericidal activity [45]. The vertically aligned MoS2 structure also has more sharp edges, which will destroy bacterial cell walls and reduce membrane integrity more efficiently [46]. The vertically aligned MoS2 structure can be decorated with other materials to further boost its antibacterial activity. Liu et al. obtained a uniform, dense and nearly vertical growth of MoS2 on the conductive support-polyaniline nanorods by a hydrothermal method [47]. These MoS2-polyaniline nanorods have large amount of active edges and perfect electron transfer efficiency, resulting in the excellent photocatalytic activity. After 3 h of visible light irradiation, the MoS2-polyaniline nanorods almost completely kill E. coli. Tang et al. vertically coated MoS2 nanosheets (with or without the doping of iron) on the titanium substrate via a one-step hydrothermal reaction. The resultant MoS2 hybrids showed satisfying antibacterial activities without the irradiation of visible light. The authors suggested that the doping of iron enhanced the bactericidal activities by releasing ferrous ions and boosting the ROS generation via Fenton-like reactions [48]. Alimohammadi et al. investigated the antimicrobial properties of the randomly oriented MnO2, MoS2 and vertically aligned MnO2/GO, MoS2/rGO, MoS2/MXene. As shown in Fig. 3c, the percentages of viable B. subtilis bacteria decreased from 95 to 60% (MoS2/rGO) and to 75% (MoS2/MXene). The vertically aligned MnO2/GO showed a remarkable bactericidal activity as the percentage of the viable bacteria decreased to 10% [46].

Due to the advances of synthetic methods, the range of vertical 2D materials has extended to other 2D materials, including TMDs [45, 49,50,51], h-BN [52], MoO3 [53], g-C3N4 [54], MXene [55] and layered double hydroxide [56, 57]. However, the vertical aligned form of other 2D materials except graphene, MnO2 and MoS2 is still scarcely investigated for bactericidal applications.

Decorating with other materials to enhance the photocatalytic activity

Photocatalysis will be initiated when the photocatalyst surface is bombarded by photons with enough energy. Irradiation of light with suitable wavelength (> the band gap of the photocatalyst) activates the electrons (e−) to escape from the valence band of the photocatalyst. When the e− leaves and is absorbed onto the conduction band (\( {\text{e}}^{ - } \)CB), a positive hole is formed on the valence band (\( {\text{h}}^{ + } \)VB) [58]. \( {\text{e}}^{ - } \)CB and \( {\text{h}}^{ + } \)VB lead to the formation of reactive species such as superoxide radicals, hydroperoxide radicals and hydroxyl radicals, which cause the microbe inactivation [59]. Therefore, the electron (e−)/hole (h+) separation and transportation to the photocatalyst surface are critical for the generation of reactive species and the following antimicrobial efficiencies.

One way to modulate the separation and transportation of e−/h+ is fabricating specific morphologies (e.g., zero-dimensional (0D) structure [60], vertically aligned structure [45], microflower structure [61]) to change the microbe inactivation effects. Another common way is to couple with foreign materials to form heterostructures, which will be discussed in this section. It has long been known that the photocatalytic performance of the single-component photocatalyst is not ideal due to the high recombination rate of photo-generated electrons and holes. Combining one photocatalyst with other materials to form the heterostructures facilitates the charge transfer between the different photocatalysts and promotes the separation of charge carriers [62, 63]. Among pg-2DMs, graphitic carbon nitride (g-C3N4) is a popular star in photocatalytic infection applications due to its broad light response spectrum (up to 460 nm), high stability and ease being fabricated and tuned to various morphologies. Yet, pristine g-C3N4 shows limited photocatalytic efficiency because of the high recombination rate of photo-generated charges and low surface area [64]. To overcome this drawback, other materials (e.g., graphene and its derivatives, noble metals) that serve as the electron sink, conductive mediator, photosensitizer and absorbent, are selected to couple with g-C3N4 and the resultant heterostructures show perfect microbial inactivation abilities. (This has been summarized by Liu et al. [65].) Apart from g-C3N4, MoS2 also gets increasing attentions in the photocatalysis area [66]. MoS2 is a semiconductor material with a narrow band gap (~ 1.8 eV) because it has a strong absorption in the visible region of solar spectrum [67]. But the fast recombination of photo-generated charges and reduced conductivity of MoS2 confine its application as the cocatalyst [68]. If it is attached with other materials such as graphene [69] and carbon [70], however, it turns out to be an efficient cocatalyst. For instance, graphene supports to improve the photocatalytic performance of MoS2 by acting as an excellent electron acceptor and transporter. Compositing MoS2 with reduced graphene (rGO) is a low-cost and efficient alternative for improving the photocatalytic activity of MoS2. The synergetic effect between MoS2 and rGO can be further boosted by the doping of ZnO, and the resultant composites display excellent photocatalytic and antibacterial activities [61]. Wang et al. combined the hydrothermal carbonation carbon (HTCC) with MoS2 to form the HTCC@MoS2 heterojunction. Compared with the single-component HTCC and MoS2, the binary composites HTCC@MoS2 exhibited super-bactericidal capacities because of their excellent photocatalytic activities (Fig. 4a) [70].

Figure 4
figure 4

a Cell density of E. coli K-12 treated with 1 mg/mL HTCC, MoS2 and HTCC@MoS2 at 37 °C for 18 h in light. (Inset represents the light and dark controls.) Reproduced with permission from Ref. [70]. Copyright 2019, ELSEVIER. b Pictures of E. coli and MRSA colony formation in the presence of Sb2Se3 nanosheets with or without the laser irradiation (808 nm, 2 W) for 5 min and followed by incubation for 24 h and 48 h. Reproduced with permission from Ref. [72]. Copyright 2019, American Chemical Society. c, d The activities of S. aureus (c) and E. coli (d) on pure Ti, PEG/MoS2–Ti, CS/PEG/MoS2–Ti, Gent and CS/Gent/PEG/MoS2–Ti without or with NIR irradiation for 5 min. The error bar indicates means ± standard deviations (n = 3): *p < 0.05, ***p < 0.001. Reproduced with permission from Ref. [78]. Copyright 2019, Royal Society of Chemistry

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Decorating with other materials to enhance the photothermal activity

Photothermal bacterial inactivation refers to hyperthermia damage to the bacterial cells using the combination of photothermal agents and light. The agents absorb the light and transfer the energy into heat through nonradiative relaxation, which will cause irreparable damage to the bacterial cells nearby [71]. For example, Miao et al. found that two-dimensional Sb2Se3 nanosheets exhibited perfect optical absorbance from the ultraviolet to the NIR region, and thus owned excellent photothermal performances. The temperature change of 300 μM Sb2Se3 nanosheets suspensions reached 29.9 °C when it was irradiated with a laser power (808 nm, 2 W). After being treated with 300 μM Sb2Se3 nanosheets under the irradiation for 5 min, the viabilities of E. coli and methicillin-resistant S. aureus (MRSA) all decreased to 0% (Fig. 4b) [72].

Some pg-2DMs (e.g., MoS2, MXenes) can be served as excellent photoabsorbing agents. Decorating with other materials can further enhance their photothermal infection performances [73, 74]. In this regard, because of their surface plasmon resonance absorption abilities, noble metals, such as gold and silver [75], are often selected to decorate pg-2DMs to achieve better photothermal conversion efficiencies (PCE). For instance, Lin et al. fabricated a kind of Ag2S@WS2 heterostructure, which exhibited strong optical absorption and high photothermal conversion efficiency. They found that pure WS2 and Ag2S@WS2 heterostructure can reach 30 °C and 60 °C, respectively, under the irradiation of NIR (808 nm) for 10 min. Pure WS2 showed a very weak antibacterial ability. In contrast, the antibacterial efficacy of Ag2S@WS2 reached up to 99.93% and 99.84% against S. aureus and E. coli, respectively [76]. Besides, in comparison with bare black phosphorus (BP) nanosheets, gold nanoparticle-decorated BP nanosheets showed 8% increase in PCE, caused by the surface-enhanced Raman scattering (SERS) of gold nanoparticles [77]. Zhang et al. decorated MoS2 nanosheets with Fe3O4 nanoparticles, which aggregated the MoS2 nanosheets and thus increase the local heat around MoS2 nanosheets during the near-infrared (NIR) irradiation, finally leading to better antibacterial activities [25].

Loading with other antibacterial agents

Pg-2DMs have large specific surface areas, making them to be perfect platforms to load small molecular antibacterial agents. Zhang et al. successfully loaded tetracycline hydrochloride drugs into the chitosan-functionalized MoS2 nanosheets. In terms of the antibacterial and antibiofilm activities, the combination of MoS2 nanosheets and antibiotics was more efficient than either working alone [7]. Moreover, the release of the loaded antibiotics can be controlled to achieve better antimicrobial efficiencies by the photothermal properties of some pg-2DMs. Ma et al. developed a photothermally modulated drug release system on the implant surface for in situ rapid disinfection. This system, called CS/Gent/PEG/MoS2–Ti, consists of chitosan (CS), gentamicin (Gent), polyethylene glycol (PEG), MoS2 and a titanium implant. When the CS/Gent/PEG/MoS2–Ti system is under the radiation of NIR, the photothermal component MoS2 increases the temperature and thus accelerates the release of Gent. Meanwhile, hyperthermia weakens the bacterial activity and enhances the membrane permeability, which helps the drugs to penetrate into the bacteria. When maintained at 50 °C for 5 min under NIR irradiation, this system can achieve infection efficiencies of 99.93% and 99.19% against E. coli and S. aureus, respectively. In contrast, even treated for 120 min by Gent alone, only a 93.79% antibacterial ratio can be obtained (Fig. 4c, d) [78].

In addition to MoS2, LDHs are often utilized as the carriers of antibacterial agents as well. The interlayer space of LDHs can be intercalated with different ions. In addition, they have good biodegradability, composition-dependent properties and are easy to be prepared, all of which make LDHs become the ideal reservoirs for sustained and controlled drug release. Thus far, numerous antibacterial agents (e.g., antibiotics [79, 80], DL-mandelic acid [81], silver nanoparticles [82], lysozyme [11, 83]) have been loaded into LDHs. (More information can be found the review contributed by Sun et al. [6].)

As the candidates for preparing novel bactericidal materials, many pg-2DMs provide new solutions to the increasingly serious bacterial infection problem. To fuel the development of this field, we would like to point out some interesting directions for the future research:

  1. (1)

    Since many pg-2DMs own large specific surface areas and lysozyme has been successfully loaded onto LDHs [11], it might be possible to fabricate more efficient and less toxic antimicrobial agents by combining the antimicrobial peptide with some suitable pg-2DMs.

  2. (2)

    Recent findings demonstrate that the phase of MoS2 can be controlled by tuning the synthesis conditions [84, 85]. Interestingly, MoS2 in 1T phase shows higher catalytic activities [86, 87], which might be utilized to generate more ROS for antimicrobial purposes. Therefore, using the methods in phase engineer to synthesize some pg-2DMs with specific phases and simultaneously decorating with foreign materials such as noble metal and metal oxides may obtain fancy antimicrobial nanocomposites.

Biosafety assessment of post-graphene 2D materials-based antimicrobial agents

While recognizing the advantages and potential huge markets of pg-2DMs in antibacterial fields, a scientific and social issue has also caught the attention of researchers—the biosafety of these pg-2DMs-AA. Biosafety, referring to the risks to the human health and environment, is the primary importance for choosing materials that are valuable to the fields like biomedicine. The biosafety of the pg-2DMs-AA being intended to be applied in the biomedicine or public health must be thoroughly evaluated prior to their practical applications. The hazard potentials of pg-2DMs themselves are not within the scope of this review because they have already been summarized by some recently published reviews [88, 89]. Here, we summarized the biosafety assessments of the antimicrobial agents fabricated by pg-2DMs or their derivatives. As shown in Fig. 5a, some researchers evaluate the biosafety of the resultant pg-2DMs-AA via in vitro assessment and/or in vivo assessment. Only a few of them explore the environmental risks of pg-2DMs-AA. Most of the prepared pg-2DMs-AA (around 70.32%) are not received any biosafety assessment. So far, the biosafety of pg-2DMs-AA fabricated by MoS2 is studied most intensively (Fig. 5b).

Figure 5
figure 5

a Percentage of the pg-2DMs-AA-associated publications with or without biosafety assessment; b percentage of different pg-2DMs-based antimicrobial agents whose biosafety has been assessed

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Assessing the risks to human health

In vitro assessment

In vitro assessment is generally performed by cell cultures, which are used as prescreening tools to unveil the toxicities of bactericidal nanomaterials and the mechanisms of action [4]. The selected cells include tumor cells (e.g., HeLa cell [8, 10, 90, 91], hepatoma cell [25], MDA-MB-231 cell (human breast cancer cell) [10, 92], KU-7 cell (urothelial carcinoma cell line) [93]) and normal cells derived from different tissues (e.g., HUVEC cell (human umbilical vein endothelial cell) [91], buffalo rat liver 3A cell [25], erythrocyte [39, 94], fibroblast cell [10, 95], human mesenchymal stem cell [10]). As summarized in Table 1, the cytotoxicities of these antimicrobial agents are often tested by colorimetric assays, which determine the variations of cell viability and proliferation (such as MTT and CCK-8 assay), and cell membrane stability (such as lactate dehydrogenase and hemolysis assay). In addition to the colorimetric assays, qRT-PCR is also utilized to detect the impacts of these pg-2DMs-AA on the gene expressions. For instance, Mao et al. have prepared a kind of black phosphorus (BP) nanosheets chitosan hydrogel to promote bacteria-accompanied wound healing. MTT assay indicates that the BP hydrogel enhances the viability of NIH-3T3 cell. The authors further investigate the expressions of smooth muscle α-actin (α-actin) and type III collagen (COL III) gene. They find the BP hydrogel facilitates the expressions of these two genes, suggesting the BP hydrogel facilitates the fibroblast cells to differentiate into myofibroblasts, and the tissue reorganization and basement membrane regeneration (Fig. 6a–d) [96].

Table 1 Biosafety assessment of the post-graphene 2D materials-based antimicrobial agents
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Figure 6
figure 6

a, b Cell viabilities of the NIH-3T3 cells exposed to tissue culture plate (TCP), chitosan (CS) or chitosan-black phosphorus (CS-BP) hydrogel without (a) or with (b) initially irradiated for 10 min, and they were continuously co-cultured in the dark for 1, 3 and 7 days afterward. c, d Expressions of smooth muscle alpha actin (α-actin) gene (c) and collagen type III (COL III) gene (d) in NIH-3T3 cells (*p < 0.05, **p < 0.01, ***p < 0.001). Reproduced with permission from Ref. [96]. Copyright 2018, American Chemical Society. e Histological images of the liver, spleen, kidney, heart and lung of the mice that were injected with the BP nanosheets, [poly(d,l-lactide)-poly(ethylene glycol)-poly(d,l-lactide) (PDLLA-PEG-PDLLA: PLEL)] hydrogel and BP@PLEL hydrogel at 20 days post-injection. Reproduced with permission from Ref. [10]. Copyright 2018, WILEY

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In vivo assessment

Bactericidal nanomaterials can penetrate into human body by the skin exposure or inhalation and then influence the major organs like lung, heart and brain. The in vivo toxicological effects of bactericidal nanomaterials are much more complicate than their in vitro effects, because the circumstances within the body are more complex than those in the cell culture medium. Thus, the results from the cell culture studies need to be confirmed by in vivo studies. Currently, in vivo assessments for pg-2DMs-AA are conducted by analyzing the histological structures of the major organs [8, 10, 31, 41, 96, 97] and/or measuring body weights of the tested animals [91, 97] (Table 1). For example, Shao et al. fabricated a kind of thermosensitive hydrogel containing black phosphorus nanosheets (BP@PLEL hydrogel). The hydrogel was subcutaneously injected into the rear parts of the Balb/c mice (6 weeks old). The mice were killed 20 days after the injection, and their major organs (liver, spleen, kidney, heart and lung) were collected and then analyzed by H&E staining. No apparent histological abnormality or lesion was observed in the mice treated with the BP@PLEL hydrogel, suggesting the hydrogel was safe within the body (Fig. 6e) [10].

Assessing the risks to environment

Nanomaterial-based antimicrobial agents widely used in human medicine, water infection or food packing will inevitably be released into the environment. They can have deleterious effects on sensitive organisms, especially the wild microorganisms, which may cause disorders in the ecosystem. Therefore, the potential impact of antimicrobial agents on the ecosystem must be evaluated as closely as other hazardous chemicals [98]. So far, the ecotoxicities of nanomaterial-based antimicrobial agents (e.g., quantum dots based [99], ZnO nanoparticle based [100]) have been explored.

However, the studies exploring the ecotoxicities of pg-2DMs-AA are still in infancy. Only two studies are found and introduced here. For instance, after exploring the antibacterial activity of Ti3C2Tx (MXene), Khaled A. Mahmoud’s group further investigated the ecotoxicity of Ti3C2Tx by evaluating its impacts on zebrafish embryos and the motor and nervous system of zebrafish larvae (Fig. 7a) [13, 101]. They found that the LC50 of Ti3C2Tx was 257.46 μg/mL. No significant teratogenic effects were observed on zebrafish embryos at 100 μg/mL. Locomotion and neurotoxicity assays further supported the nontoxicity of Ti3C2Tx at a concentration of 50 μg/mL. Rozmysłowska et al. examined how the modifications of the antimicrobial Ti3C2 MXene with ceramic oxide and noble metal nanoparticles affect the corresponding ecotoxicities. Three nanocomposites (Ti3C2/3% Al2O3/2% Ag, Ti3C2/3% SiO2/2% Ag and Ti3C2/3% SiO2/2% Pd) were synthesized, and their impacts on the green algae (Desmodesmus quadricauda), two species of higher plants [sorghum (Sorghum saccharatum) and charlock (Sinapis alba)] were explored (Fig. 7b–f). The authors reported that the modification of Ti3C2 MXene with Al2O3 + Ag, SiO2 + Ag and SiO2 + Pd resulted in significant increases of antibacterial activities. The resultant nanomaterials caused both stimulating and inhibiting effects toward algae, which were depended on the concentration and the type of components. Yet, all the modifications resulted in a decrease in phytotoxicity toward the two plants in terms of sprout. The root growth of the plants was inhibited in the absence of Ti3C2/Al2O3/Ag composites. The strongest inhibition of the seed germination (up to 40%) was observed in the group treated with Ti3C2 MXene [102].

Figure 7
figure 7

a Acute toxicity assessment of dimethyl sulfoxide (DMSO), diethylaminobenzaldehyde (DEAB) and Ti3C2Tx (MXene). Reproduced with permission from Ref. [101]. Copyright 2018, Royal Society of Chemistry. b Impacts of pristine Ti3C2MXene, Ti3C2/Al2O3/Ag, Ti3C2/SiO2/Ag and Ti3C2/SiO2/Pd nanocomposite on the algal growth after the incubation of 2 weeks. c–f The impacts of different nanopowders (100 mg/kg, 200 mg/kg) on the sprout length (c, e) and root length (d, f) of Sorghum saccharatum and Sinapis alba. Reproduced with permission from Ref. [102]. Copyright 2019, Royal Society of Chemistry

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As demonstrated above, the biosafety assessments of pg-2DMs-AA for the environmental safety or human health are summarized. However, more efforts are still needed in this direction and the following suggestions are given:

  1. (1)

    Currently, dye-based colorimetric assays such as MTT assay are often utilized to determine the variations of cell viability in the in vitro assessment of pg-2DMs-AA. Yet, some reports suggest that MTT dyes might react with pg-2DMs such as MoS2 and black phosphorous, causing perturbations to the final results [103, 104]. Therefore, to ensure the accuracies of assessment results, other approaches such as the electrical impedance spectroscopy can be utilized and compared with the colorimetric assays [105].

  2. (2)

    The characteristics of the selected cells should be considered thoroughly for in vitro cytotoxicity assessment of pg-2DMs-AA. Different cell lines show different sensitivities to 2D material exposure [106, 107]. It has been shown that tumor cells are less sensitive to nanomaterials since they are more robust than the normal cells after all [108]. Therefore, we should be very careful in understanding the in vitro cytotoxicity results of pg-2DMs-AA which are based on some tumor cells such as HeLa cell.

  3. (3)

    Cells response sensitively to the external stimulus at the molecular level and increasing reports explore the molecular events to fully evaluate the toxicities of nanomaterials [109, 110]. It will be more sensitive to decipher the biosafety of pg-2DMs-AA from RNA, protein and metabolite levels, which will be further supported by the high-throughput omics technologies [111].

  4. (4)

    Little is known regarding the ecotoxicities of pg-2DMs-AA. As we known, the ecotoxicities of nanomaterials highly depend on their own physiochemical properties (e.g., size, thickness and component) [112]. Some insights regarding the ecotoxicities of pg-2DMs have already been obtained. Given that pg-2DMs-AA are quite different from their parent pg-2DMs in terms of physiochemical properties, the ecotoxicities of pg-2DMs-AA will be varied from those of pg-2DMs, and thus more efforts are needed in this direction.

Conclusion and perspective

Antibiotic-resistant bacteria have become a serious public health problem, causing a strong demand to develop novel agents with superior antimicrobial activities. The emergence of pg-2DMs with unique properties provides new alternatives to prepare antimicrobial agents. In this review, we summarized recent advances in the methods for boosting the antimicrobial activities of pg-2DMs-AA and evaluating the biosafety of pg-2DMs-AA. More excellent antimicrobial agents can be obtained based on pg-2DMs by incorporating more new approaches such as phase engineer. Yet, the safety assessment should not be ignored as we expand the applications of pg-2DMs-AA. More efforts are needed in this aspect, especially the ecological safety evaluation. All in all, we believe pg-2DMs will provide more powerful weapons for the fight against pathogens.